RESUMEN
Blood-borne RNA circulating in association with autoantibodies is a potent stimulator of interferon production and immune system activation. RSLV-132 is a novel fully human biologic Fc fusion protein that is comprised of human RNase fused to the Fc domain of human IgG1. The drug is designed to remain in circulation and digest extracellular RNA with the aim of preventing activation of the immune system via Toll-like receptors and the interferon pathway. The present study describes the first clinical study of nuclease therapy in 32 subjects with systemic lupus erythematosus. The drug was well tolerated with a very favorable safety profile. The approximately 19-day serum half-life potentially supports once monthly dosing. There were no subjects in the study that developed anti-RSLV-132 antibodies. Decreases in B-cell activating factor correlated with decreases in disease activity in a subset of patients.
Asunto(s)
Autoanticuerpos/sangre , Lupus Eritematoso Sistémico/tratamiento farmacológico , ARN/sangre , Proteínas Recombinantes de Fusión/uso terapéutico , Adulto , Autoanticuerpos/inmunología , Factor Activador de Células B/metabolismo , Método Doble Ciego , Esquema de Medicación , Femenino , Semivida , Humanos , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Ribonucleasas/inmunología , Índice de Severidad de la EnfermedadRESUMEN
To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Proteínas Reguladoras de la Apoptosis , Relación Dosis-Respuesta a Droga , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/síntesis química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Conformación Proteica , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/síntesis químicaRESUMEN
TRAIL-R3, a new member of the TRAIL receptor family, has been cloned and characterized. TRAIL-R3 encodes a 299 amino acid protein with 58 and 54% overall identity to TRAIL-R1 and -R2, respectively. Transient expression and quantitative binding studies show TRAIL-R3 to be a plasma membrane-bound protein capable of high affinity interaction with the TRAIL ligand. The TRAIL-R3 gene maps to human chromosome 8p22-21, clustered with the genes encoding two other TRAIL receptors. In contrast to TRAIL-R1 and -R2, this receptor shows restricted expression, with transcripts detectable only in peripheral blood lymphocytes and spleen. The structure of TRAIL-R3 is unique when compared to the other TRAIL receptors in that it lacks a cytoplasmic domain and appears to be glycosyl-phosphatidylinositol-linked. Moreover, unlike TRAIL-R1 and -R2, in a transient overexpression system TRAIL-R3 does not induce apoptosis.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 8/genética , Clonación Molecular , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Glicosilfosfatidilinositoles , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Células Jurkat , Linfocitos/química , Linfocitos/metabolismo , Glicoproteínas de Membrana/farmacología , Datos de Secuencia Molecular , Unión Proteica , Receptores de Superficie Celular/genética , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/farmacología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ligando Inductor de Apoptosis Relacionado con TNF , Receptores Señuelo del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-A resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a beta-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a beta-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.
Asunto(s)
Virus de la Viruela Vacuna/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Células CHO , Quimiocinas/antagonistas & inhibidores , Quimiocinas/metabolismo , Cricetinae , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/metabolismoRESUMEN
We show the cowpox genome (Brighton Red strain) contains a single copy gene, crmC, expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The approximately 25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LT alpha, TNF beta) and lacks the conserved (but nonhomologous) approximately 150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF.
Asunto(s)
Virus de la Viruela Vacuna/genética , Receptores del Factor de Necrosis Tumoral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Virus de la Viruela Vacuna/fisiología , ADN Viral , Genes Virales , Genoma Viral , Humanos , Linfotoxina-alfa/metabolismo , Ratones , Datos de Secuencia Molecular , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/metabolismo , Homología de Secuencia de Aminoácido , Solubilidad , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
The lymphotoxin beta receptor (LT beta R) was originally described as a transcribed sequence encoded on human chromosome 12p, with homology to the TNF receptor family. Subsequently, a recombinant LT beta R was shown to bind LT alpha LT beta heteromeric complexes. In this study, we have shown that LT beta R is expressed in a variety of tissues and cell lines of monocytic lineage, as well as in fibroblast and human melanoma cell lines. Unlike other members of the TNF receptor family, LT beta R is not expressed by peripheral blood T cells. A chimeric fusion protein consisting of the extracellular domain of LT beta R fused to the Fc region of human IgG1 was used to develop mAbs against LT beta R. Cross-linking LT beta R on A375 melanoma cells with these Abs generated an antiproliferative signal. In addition, the IL-8 and RANTES chemokines, early indicators of inflammation, were secreted by the A375 melanoma line and the WI38VA13 fibroblast line in response to cross-linking of LT beta R. These same activities could be induced by membrane-bound and soluble LT beta and LT alpha LT beta oligomers.
Asunto(s)
Quimiocinas/biosíntesis , Inhibidores de Crecimiento/fisiología , Linfotoxina-alfa/metabolismo , Linfotoxina-alfa/fisiología , Melanoma/inmunología , Melanoma/patología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Adulto , Anticuerpos Monoclonales/biosíntesis , Biopolímeros , Reactivos de Enlaces Cruzados , Feto , Inhibidores de Crecimiento/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Receptor beta de Linfotoxina , Linfotoxina-alfa/inmunología , Linfotoxina beta , Melanoma/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/farmacología , Receptores del Factor de Necrosis Tumoral/inmunología , Solubilidad , Distribución Tisular , Células Tumorales CultivadasRESUMEN
A fourth member of the emerging TRAIL receptor family, TRAIL-R4, has been cloned and characterized. TRAIL-R4 encodes a 386-amino acid protein with an extracellular domain showing 58%-70% identity to those of TRAIL-R1, TRAIL-R2, and TRAIL-R3. The signaling capacity of TRAIL-R4 is similar to that of TRAIL-R1 and TRAIL-R2 with respect to NF-kappaB activation, but differs in its inability to induce apoptosis. Yet TRAIL-R4 retains a C-terminal element containing one third of a consensus death domain motif. Transient overexpression of TRAIL-R4 in cells normally sensitive to TRAIL-mediated killing confers complete protection, suggesting that one function of TRAIL-R4 may be inhibition of TRAIL cytotoxicity. Like TRAIL-R1 and TRAIL-R2, this receptor shows widespread tissue expression. The human TRAIL-R4 gene has been mapped to chromosome 8p22-21, clustered with three other TRAIL receptors.
Asunto(s)
Apoptosis , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Sitios de Unión , ADN Complementario , Humanos , Células Jurkat , Datos de Secuencia Molecular , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/genética , Homología de Secuencia de Aminoácido , Ligando Inductor de Apoptosis Relacionado con TNF , Distribución TisularRESUMEN
TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Glicoproteínas de Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Linfocitos B/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 8 , Clonación Molecular , Cisteína Endopeptidasas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/aislamiento & purificación , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/farmacología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF , Distribución TisularRESUMEN
Poxvirus genomes encode several proteins which inhibit specific elements of the host immune response. We show the "35K" virulence gene in variola and cowpox viruses, whose vaccinia and Shope fibroma virus equivalents are strongly conserved in sequence, actually encodes a secreted soluble protein with high-affinity binding to virtually all known beta chemokines, but only weak or no affinity to the alpha and gamma classes. The viral protein completely inhibits the biological activity of monocyte chemotactic protein-1 (MCP-1) by competitive inhibition of chemokine binding to cellular receptors. As all beta chemokines are also shown to cross-compete with MCP1 binding to the viral protein, we conclude that this viral chemokine inhibitor (vCCI) not only interacts through a common binding site, but is likely a potent general inhibitor of beta chemokine activity. Unlike many poxvirus virulence genes to date, which are clearly altered forms of acquired cellular genes of the vertebrate immune system, this viral chemokine inhibitor (vCCI) shares no sequence homology with known proteins, including known cellular chemokine receptors, all of which are multiple membrane-spanning proteins. Thus, vCCI presumably has no cellular analogue and instead may be the product of unrelenting sequence variations which gave rise to a completely new protein with similar binding properties to native chemokine receptors. The proposed function of vCCI is inhibition of the proinflammatory (antiviral) activities of beta chemokines.
Asunto(s)
Quimiocinas/antagonistas & inhibidores , Genoma Viral , Poxviridae/genética , Poxviridae/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Calcio/metabolismo , Línea Celular , Quimiocinas/clasificación , Quimiotaxis de Leucocito , Quimera/genética , Quimera/inmunología , Virus de la Viruela Vacuna/genética , Virus de la Viruela Vacuna/inmunología , Cartilla de ADN/genética , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Quimiocina/genética , Homología de Secuencia de Aminoácido , Solubilidad , Virus de la Viruela/genética , Virus de la Viruela/inmunología , Proteínas Virales/metabolismo , Virulencia/genéticaRESUMEN
A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.
Asunto(s)
Apoptosis/efectos de los fármacos , Cromosomas Humanos Par 3 , ADN Complementario/aislamiento & purificación , Glicoproteínas de Membrana/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Humanos , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/aislamiento & purificación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.