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Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.
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Anticuerpos Biespecíficos , Antígeno CD47 , Antígeno Carcinoembrionario , Animales , Femenino , Humanos , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Antígeno CD47/inmunología , Antígeno CD47/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas Ligadas a GPI , Macaca fascicularis , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. RESULTS: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).
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The aberrant release of inflammatory mediators often referred to as a cytokine storm or cytokine release syndrome (CRS), is a common and sometimes fatal complication in acute infectious diseases including Ebola, dengue, COVID-19, and influenza. Fatal CRS occurrences have also plagued the development of highly promising cancer therapies based on T-cell engagers and chimeric antigen receptor (CAR) T cells. CRS is intimately linked with dysregulated and excessive cytokine release, including IFN-γ, TNF-α, IL 1, IL-6, and IL-10, resulting in a systemic inflammatory response leading to multiple organ failure. Here, we show that mice intravenously administered the agonistic hamster anti-mouse CD3ε monoclonal antibody 145-2C11 develop clinical and laboratory manifestations seen in patients afflicted with CRS, including body weight loss, hepatosplenomegaly, thrombocytopenia, increased vascular permeability, lung inflammation, and hypercytokinemia. Blood cytokine levels and gene expression analysis from lung, liver, and spleen demonstrated a hierarchy of inflammatory cytokine production and infiltrating immune cells with differentiating organ-dependent kinetics. IL-2, IFN-γ, TNF-α, and IL-6 up-regulation preceded clinical signs of CRS. The co-treatment of mice with a neutralizing anti-cytokine antibody cocktail transiently improved early clinical and laboratory features of CRS. We discuss the predictive use of this model in the context of new anti-cytokine strategies to treat human CRS.
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Anticuerpos Monoclonales/farmacología , Anticuerpos/inmunología , Complejo CD3/antagonistas & inhibidores , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/metabolismo , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Animales , Anticuerpos/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Síndrome de Liberación de Citoquinas/diagnóstico , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Citocinas/sangre , Modelos Animales de Enfermedad , Quimioterapia Combinada , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Ratones , Fenotipo , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in several solid malignancies, including gastric, lung, mesothelioma, pancreatic and ovarian cancers. While several MSLN-targeting therapeutic approaches are in development, only limited efficacy has been achieved in patients. A potential shortcoming of several described antibody-based approaches is that they target the membrane distal region of MSLN and, additionally, are known to be handicapped by the high levels of circulating soluble MSLN in patients. We show here, using monoclonal antibodies (mAbs) targeting different MSLN-spanning epitopes, that the membrane-proximal region resulted in more efficient killing of MSLN-positive tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Surprisingly, no augmented killing was observed in antibody-dependent cellular phagocytosis (ADCP) by mAbs targeting this membrane-proximal region. To further increase the ADCP potential, we, therefore, generated bispecific antibodies (bsAbs) coupling a high-affinity MSLN binding arm to a blocking CD47 arm. Here, targeting the membrane-proximal domain of MSLN demonstrated enhanced ADCP activity compared to membrane-distal domains when the bsAbs were used in in vitro phagocytosis killing assays. Importantly, the superior anti-tumor activity was also translated in xenograft tumor models. Furthermore, we show that the bsAb approach targeting the membrane-proximal epitope of MSLN optimized ADCC activity by augmenting FcγR-IIIA activation and enhanced ADCP via a more efficient blockade of the CD47/SIRPα axis.
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Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Antígeno CD47/inmunología , Proteínas Ligadas a GPI/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/farmacología , Epítopos/inmunología , Humanos , Inmunoterapia/métodos , Mesotelina , Ratones , Fagocitosis/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 vs. 0 pg/mL, P=0.03, and 1514.0±773 vs. 233.6±50.1 pg/mlL, P=0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1-/-mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8+ cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.
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Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Interferón gamma/metabolismo , Adolescente , Animales , Biopsia , Médula Ósea/metabolismo , Médula Ósea/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Niño , Preescolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/etiología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunohistoquímica , Memoria Inmunológica , Lactante , Masculino , Ratones , Ratones Noqueados , Donantes de Tejidos , Trasplante Homólogo , Adulto JovenRESUMEN
CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.
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Anticuerpos Biespecíficos/farmacología , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Antígeno CD47/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Activación de Linfocitos/efectos de los fármacosRESUMEN
CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20-targeted therapy. Mol Cancer Ther; 17(8); 1739-51. ©2018 AACR.
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Anticuerpos Biespecíficos/genética , Leucemia/genética , Leucemia/terapia , Linfoma de Células B/genética , Linfoma de Células B/terapia , Animales , Antígenos CD19 , Antígeno CD47 , Humanos , Leucemia/patología , Linfoma de Células B/patología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10-60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Quimiocina CXCL10/antagonistas & inhibidores , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Endotelio Vascular/metabolismo , Glicosaminoglicanos/metabolismo , Animales , Anticuerpos Monoclonales/farmacocinética , Células Cultivadas , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiotaxis de Leucocito/fisiología , Cricetinae , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Distribución TisularRESUMEN
Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.
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Interferón gamma/sangre , Linfohistiocitosis Hemofagocítica/sangre , Animales , Biomarcadores/sangre , Quimiocina CXCL10/sangre , Quimiocina CXCL9/sangre , Niño , Modelos Animales de Enfermedad , Femenino , Humanos , Linfohistiocitosis Hemofagocítica/patología , Ratones Endogámicos C57BL , Pruebas de Neutralización , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , SíndromeRESUMEN
The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.
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Interleucina-6/química , Interleucina-6/metabolismo , Receptores de Interleucina-6/química , Receptores de Interleucina-6/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Femenino , Prueba de Complementación Genética , Humanos , Interleucina-6/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células 3T3 NIH , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ratas , Receptores de Interleucina-6/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
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
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
B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.
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Enfermedades Autoinmunes/prevención & control , Linfocitos B/inmunología , Antígenos CD79/inmunología , Anergia Clonal/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Autoinmunidad/inmunología , Femenino , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones NoqueadosRESUMEN
Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.
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Anticuerpos/genética , Animales , Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Biblioteca de PéptidosRESUMEN
AIMS: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS: C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION: Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.
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Anticuerpos Monoclonales/farmacología , Quimiocina CCL5/fisiología , Insuficiencia Cardíaca/patología , Infarto del Miocardio/patología , Animales , Peso Corporal/fisiología , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/inmunología , Quimiotaxis de Leucocito/fisiología , Enfermedad Crónica , Colágeno/metabolismo , Ligadura , Macrófagos/fisiología , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología , Isquemia Miocárdica/metabolismo , Reperfusión Miocárdica/métodos , Infiltración Neutrófila/fisiología , Neutrófilos/fisiología , Tamaño de los Órganos/fisiología , Tasa de SupervivenciaRESUMEN
RATIONALE: The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients. OBJECTIVES: To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA. METHODS: We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure. MEASUREMENTS AND MAIN RESULTS: IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia. CONCLUSIONS: Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.
Asunto(s)
Aterosclerosis/metabolismo , Quimiocina CCL5/metabolismo , Hipoxia/metabolismo , Inflamación/metabolismo , Síndromes de la Apnea del Sueño/metabolismo , Animales , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Quimiocina CCL5/genética , Modelos Animales de Enfermedad , Hipoxia/genética , Hipoxia/patología , Inflamación/genética , Masculino , Arterias Mesentéricas/patología , Ratones , Ratones Endogámicos C57BL , Microscopía , Síndromes de la Apnea del Sueño/genética , Transcripción Genética/genética , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.
Asunto(s)
Pulmón/inmunología , Pulmón/patología , Neumonía/inmunología , Receptores de Quimiocina/metabolismo , Células Th2/citología , Células Th2/inmunología , Animales , Antígenos de Protozoos/inmunología , Apoptosis , Hiperreactividad Bronquial/complicaciones , Hiperreactividad Bronquial/inmunología , Receptor 1 de Quimiocinas CX3C , Proliferación Celular , Supervivencia Celular , Hipersensibilidad/complicaciones , Hipersensibilidad/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Transgénicos , Fenotipo , Neumonía/complicaciones , Proteínas Protozoarias/inmunología , Receptores de Interleucina-8A/metabolismo , Transducción de SeñalRESUMEN
IL-6-mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6-mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.
Asunto(s)
Artritis Experimental/inmunología , Autoinmunidad/inmunología , Interleucina-6/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Artritis Experimental/metabolismo , Separación Celular , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Interleucina-6/metabolismo , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores de Interleucina-6/inmunología , Receptores de Interleucina-6/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
Chemokines are key regulators of leukocyte trafficking and play a crucial role under homeostatic and inflammatory conditions. Because chemokines are involved in multiple pathologies, they represent an attractive class of therapeutic targets. However, because of the redundancy of this system, neutralizing a single chemokine may be insufficient to achieve therapeutic benefit. Our strategy was to use a Fc-fusion recombinant protein form of the poxvirus-derived viral CC chemokine inhibitor protein (vCCI-Fc) that has the ability to specifically bind to multiple CC chemokines and neutralize their activity. In this study, we demonstrate first that, in vivo, vCCI-Fc prevents CC chemokine-dependent migration of macrophages into inflamed tissue of carageenan-challenged mice. We next studied this effect of inhibiting CC chemokine activity in a model more relevant to human disease, collagen-induced arthritis. Mice receiving vCCI-Fc revealed a striking retention of splenocytes, including activated and IFN-gamma-secreting CD4(+) and CD8(+) T cells, that was associated with a concomitant decrease of cells in the draining lymph nodes. These phenomena resulted in a significant decrease in the incidence of disease and a reduction in clinical score, joint inflammation, and cartilage destruction as compared with mice receiving isotype control. Taken together, these results define a role for CC chemokines in the control of disease, as interfering with their function leads to a previously unappreciated role of controlling inflammatory cell trafficking in and out of secondary lymphoid organs.
Asunto(s)
Artritis Experimental/inmunología , Quimiocinas CC/inmunología , Inflamación/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Artritis Experimental/prevención & control , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/metabolismo , Femenino , Citometría de Flujo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inflamación/prevención & control , Interferón gamma/inmunología , Interferón gamma/metabolismo , Recuento de Linfocitos , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
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.