RESUMEN
BACKGROUND: Food allergies are common and are associated with substantial morbidity; the only approved treatment is oral immunotherapy for peanut allergy. METHODS: In this trial, we assessed whether omalizumab, a monoclonal anti-IgE antibody, would be effective and safe as monotherapy in patients with multiple food allergies. Persons 1 to 55 years of age who were allergic to peanuts and at least two other trial-specified foods (cashew, milk, egg, walnut, wheat, and hazelnut) were screened. Inclusion required a reaction to a food challenge of 100 mg or less of peanut protein and 300 mg or less of the two other foods. Participants were randomly assigned, in a 2:1 ratio, to receive omalizumab or placebo administered subcutaneously (with the dose based on weight and IgE levels) every 2 to 4 weeks for 16 to 20 weeks, after which the challenges were repeated. The primary end point was ingestion of peanut protein in a single dose of 600 mg or more without dose-limiting symptoms. The three key secondary end points were the consumption of cashew, of milk, and of egg in single doses of at least 1000 mg each without dose-limiting symptoms. The first 60 participants (59 of whom were children or adolescents) who completed this first stage were enrolled in a 24-week open-label extension. RESULTS: Of the 462 persons who were screened, 180 underwent randomization. The analysis population consisted of the 177 children and adolescents (1 to 17 years of age). A total of 79 of the 118 participants (67%) receiving omalizumab met the primary end-point criteria, as compared with 4 of the 59 participants (7%) receiving placebo (P<0.001). Results for the key secondary end points were consistent with those of the primary end point (cashew, 41% vs. 3%; milk, 66% vs. 10%; egg, 67% vs. 0%; P<0.001 for all comparisons). Safety end points did not differ between the groups, aside from more injection-site reactions in the omalizumab group. CONCLUSIONS: In persons as young as 1 year of age with multiple food allergies, omalizumab treatment for 16 weeks was superior to placebo in increasing the reaction threshold for peanut and other common food allergens. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT03881696.).
Asunto(s)
Antialérgicos , Desensibilización Inmunológica , Hipersensibilidad a los Alimentos , Omalizumab , Adolescente , Niño , Humanos , Lactante , Alérgenos/efectos adversos , Arachis/efectos adversos , Desensibilización Inmunológica/métodos , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/tratamiento farmacológico , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/terapia , Omalizumab/efectos adversos , Omalizumab/uso terapéutico , Hipersensibilidad al Cacahuete/tratamiento farmacológico , Hipersensibilidad al Cacahuete/inmunología , Hipersensibilidad al Cacahuete/terapia , Antialérgicos/administración & dosificación , Antialérgicos/uso terapéutico , Preescolar , Adulto Joven , Adulto , Persona de Mediana EdadRESUMEN
Background: Food allergy is common and causes substantial morbidity and even mortality. Safe and effective treatments for food allergy would therefore be highly desirable, especially for individuals with multiple food allergies. Objectives: Our aim was to describe a phase 3 study on treatment of patients with multiple food allergies with omalizumab. Methods: The study was developed as a collaboration between the Consortium for Food Allergy Research, the National Institute of Allergy and Infectious Diseases, and 2 industry sponsors (Genentech and Novartis). Results: The study is currently under way, enrolling participants from age 1 year to age 55 years who are allergic to peanut and at least 2 other foods (including milk, egg, wheat, cashew, hazelnut, and walnut). The study is designed to address 3 major questions. First, stage 1 will study the potential value of omalizumab for the treatment of patients with peanut allergy and at least 2 other common food allergens. Second, stage 2 will directly compare treatment of patients with multifood allergies using omalizumab as monotherapy versus treatment with omalizumab-facilitated multiallergen oral immunotherapy in which omalizumab is used as an adjunctive treatment. Third, stage 3 will address the longer-term outcomes following these treatment approaches, including the introduction of dietary forms of the study foods to induce or maintain desensitization. Conclusions: This phase 3 study will provide important information on the potential of omalizumab to treat patients with multiple food allergies.
RESUMEN
We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell-killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19(-) cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Linfocitos B/inmunología , Linfoma de Células B , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linfocitos B/citología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Comunicación Celular/inmunología , Línea Celular Tumoral , Femenino , Humanos , Linfocinas/inmunología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Sialoglicoproteínas/inmunología , Linfocitos T/citología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. METHODS: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. RESULTS: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. CONCLUSION: This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.
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Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Linfocitos B/efectos de los fármacos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptores de IgG , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Antígenos CD79/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dimerización , Femenino , Humanos , Inmunoglobulinas/metabolismo , Inmunosupresores/inmunología , Inmunosupresores/farmacocinética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Receptores de IgG/inmunología , Transducción de Señal , Bazo/citología , Andamios del TejidoRESUMEN
The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcgammaRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcgamma receptor (FcgammaR)-mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1). FcgammaR-mediated STAT1 activation is rapid and requires activating FcgammaRs. However, this IFN response is observed without a detectable increase in the expression of type I IFNs themselves or the need to add exogenous IFNs. Induction of IFN response genes plays an important role in FcgammaR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcgammaR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcgammaRs may regulate IFN signaling in myeloid cells. Manipulation of FcgammaR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer.
Asunto(s)
Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Interferón Tipo I/inmunología , Monocitos/inmunología , Receptores de IgG/antagonistas & inhibidores , Transducción de Señal/inmunología , Anticuerpos/farmacología , Western Blotting , Citocinas/metabolismo , Células Dendríticas/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Interferón Tipo I/metabolismo , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Factor de Transcripción STAT1/genéticaRESUMEN
Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/sangre , Receptores de IgG/sangre , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos CD/genética , Antígenos CD/inmunología , Genotipo , Humanos , Leucocitos/inmunología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Receptores de IgG/genética , Receptores de IgG/inmunología , Alineación de Secuencia , Transducción de Señal/inmunologíaRESUMEN
Human CD32B (FcgammaRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (mAb 2B6), from a novel panel of anti-human CD32B-specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5 were capable of directing cytotoxicity by peripheral blood mononuclear cells and monocyte-derived macrophages against B-lymphoma lines in vitro. In a human B-cell lymphoma mouse xenograft model, administration of ch2B6 or hu2B6-3.5 reduced tumor growth rate and improved tumor-free survival. Both the in vitro and in vivo activities of 2B6 required an intact Fc, suggesting an FcR-mediated mechanism of action. These data support the hypothesis that CD32B is a viable target for mAb treatment of B-cell lymphoproliferative disorders.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD/biosíntesis , Antígenos CD/química , Linfoma de Células B/metabolismo , Linfoma de Células B/terapia , Receptores de IgG/biosíntesis , Receptores de IgG/química , Animales , Linfocitos B/metabolismo , Células CHO , Cricetinae , Supervivencia sin Enfermedad , Humanos , Inmunohistoquímica , Linfoma de Células B/inmunología , Macrófagos/metabolismo , Ratones , Metástasis de la Neoplasia , Trasplante de NeoplasiasRESUMEN
Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcgamma receptor (FcgammaR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcgammaRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcgammaRs on human DCs to generate either antigen-specific immunity or tolerance.
Asunto(s)
Antígenos CD/inmunología , Células Dendríticas/metabolismo , Inmunoglobulina G/inmunología , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Receptores de IgG/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Relación Dosis-Respuesta Inmunológica , Humanos , Enfermedades del Complejo Inmune/inmunología , Enfermedades del Complejo Inmune/fisiopatología , Enfermedades del Complejo Inmune/terapia , Inmunoglobulina G/farmacología , Inflamación/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Isoantígenos/inmunología , Activación de Linfocitos , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos TRESUMEN
Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.
Asunto(s)
Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Bovinos , Línea Celular , Proteínas de Unión al ADN/metabolismo , Diglicéridos/fisiología , Humanos , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFATC , Proteínas Nucleares/metabolismo , Fosfolipasa C gamma , Fosforilación , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Transfección , Fosfolipasas de Tipo C/deficiencia , Fosfolipasas de Tipo C/genética , Tirosina/genéticaRESUMEN
We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1. Additional data indicate that the SH3 domain of PLCgamma1 binds to both canonical and noncanonical SH3 domain-binding sites in the proline-rich region of c-Cbl. Overexpression of c-Cbl in a PLCgamma-deficient B cell line, P10-14, stably reconstituted with wild-type PLCgamma1 led to a significant decrease in B cell receptor-induced NF-AT-dependent transcription, a PLCgamma- and Ca(2+)-dependent event. In contrast, c-Cbl overexpression in P10-14 cells reconstituted with a PLCgamma1 SH3 domain mutant had little effect on receptor-induced NF-AT activation. These data suggest that c-Cbl-mediated regulation of PLCgamma1 requires an interaction between c-Cbl and PLCgamma1 that is primarily mediated by the SH3 domain of PLCgamma1. The interaction of c-Cbl with PLCgamma1 may negatively effect events required for PLCgamma1 activation.
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Linfocitos/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Antígenos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Ubiquitina-Proteína Ligasas , Dominios Homologos src/inmunología , Animales , Sitios de Unión/inmunología , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/inmunología , Retroalimentación Fisiológica/inmunología , Humanos , Células Jurkat , Linfocitos/inmunología , Mutación/genética , Factores de Transcripción NFATC , Fosfolipasa C gamma , Fosforilación , Prolina/inmunología , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-cbl , Receptores de Antígenos/inmunología , Factores de Transcripción/metabolismo , Activación Transcripcional/inmunología , Fosfolipasas de Tipo C/inmunología , Tirosina/metabolismoAsunto(s)
Linfocitos B/inmunología , Linfocitos T/inmunología , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Dominio Catalítico , Regulación Enzimológica de la Expresión Génica , Humanos , Microdominios de Membrana , Modelos Biológicos , Fosfolipasa C gamma , Fosforilación , Estructura Terciaria de Proteína , Transducción de Señal , Tirosina/metabolismoRESUMEN
The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase C gamma 1 (PLC gamma 1) activation. 70Z/3 Cbl-induced PLC gamma 1 tyrosine phosphorylation required, in addition to the PLC gamma 1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation. Deletion of the leucine zipper of 70Z/3 Cbl did not eliminate 70Z/3 Cbl-induced PLC gamma 1 phosphorylation, suggesting that blockage of c-Cbl via dimerization with 70Z/3 Cbl cannot fully explain 70Z/3 Cbl activating characteristics. The complete elimination of PLC gamma 1 phosphorylation required deleting the SH3 domain-binding region of 70Z/3 Cbl, consistent with 70Z/3 Cbl binding the PLC gamma 1 SH3 domain. 70Z/3 Cbl-induced PLC gamma 1 phosphorylation required Zap-70, as for the TCR, and the tyrosine kinase binding domain of 70Z/3 Cbl, which binds Zap-70, but did not require PLC gamma 1 binding to Lat, a crucial interaction in TCR-induced PLC gamma 1 phosphorylation. Furthermore, 70Z/3 Cbl-induced activation of NFAT, a PLC gamma 1/Ca(2+)-dependent transcriptional event, required Zap-70, but was independent of Slp-76, an adapter required for TCR-induced NFAT activation. These results suggest that 70Z/3 Cbl and PLC gamma 1 form a TCR-, Lat- and Slp-76-independent complex that leads to PLC gamma 1 phosphorylation and activation.