RESUMEN
The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Neoplasias/inmunología , Radioinmunoterapia/métodos , Seroglobulinas/inmunología , Anticuerpos Monoclonales/efectos adversos , Humanos , Neoplasias/radioterapia , Radioinmunoterapia/efectos adversosRESUMEN
The sixth complement component (C6) is an essential component of the biologically active C5b-9 membrane attack complex of the complement system. The multimolecular C5b-9 complex is an important mediator of the biological effects of the activated complement system through its prominent cell signaling and cytolytic functions. To begin to provide essential information and reagents needed to analyze the functions of the complement system in mouse models of human diseases, the cDNA of the A form of mouse C6, which is present in all mouse strains, was cloned and characterized structurally and functionally. Although strikingly homologous in deduced amino acid sequence and modular structure to human C6 (75% identity), mouse C6 is substantially smaller due to the absence of the two carboxyl-terminal factor I modules (FIMs) found in human C6. Various approaches, including studies with antibody generated to recombinant mouse C6, failed to reveal evidence for FIMs in this form of mouse C6. Despite the absence of these modules in C6A, reported to be important for interactions with C5 in the human system, mouse C6A is functionally active and is readily incorporated into the mouse C5b-9 complex.
Asunto(s)
Complemento C6/genética , Factor I de Complemento/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Clonación Molecular , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , ADN Complementario , Humanos , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de AminoácidoRESUMEN
We hypothesize that amyloid (Abeta) peptide-containing neuritic plaques in the brains of patients with Alzheimer's disease represent chronic inflammatory foci mediated by the actions of the complement system and proinflammatory cytokines. In support of this, in vitro studies show that the (Abeta) peptide is a potent complement activator and that such complement activation leads to the formation of covalent (Abeta)-C3 activation fragment complexes, the generation of the chemokine-like C5a complement activation peptide, and the formation of the proinflammatory C5b-9 complex in functionally active form able to insert into neuronal cell membranes. Other studies show that C5a, together with (Abeta), synergistically augments the release of proinflammatory cytokines from human monocytes. These studies, which provide in vitro support for the hypothesis, are being pursued in an animal model of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Placa Amiloide/inmunología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Humanos , Inflamación/inmunología , Inflamación/patologíaRESUMEN
Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Complemento C3/metabolismo , Complemento C3b/metabolismo , Complemento C5/metabolismo , Complemento C5a/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Complemento C5b , Humanos , ConejosRESUMEN
An early step in the initiation of the classical C pathway is the proteolytic activation of component C4 by subcomponent C1-s. We have examined the substrate specificity of murine C1-s (mC1-s) by measuring its proteolytic activity on human and murine C4, and on the murine C4 isotype designated sex-limited protein (Slp). The latter substrate was examined because previous studies have demonstrated that Slp is not cleaved by C1-s, and hence Slp has been assumed to be nonfunctional in the C pathways. Those earlier studies used human, not murine, C1-s, however; a recent report has suggested that Slp is an essential component of a novel complement activation pathway and that the previous failure to observe cleavage of Slp is probably the result of a species incompatibility between Slp and the heterologous human C1-s (hC1-s). The present studies do not support this idea, as we found no evidence of cleavage of Slp by homologous murine C1-s even at enzyme concentrations 10-fold higher than that necessary for 50% cleavage of murine C4 (mC4). We did find a species-specific affect in the cleavage of mC4, where mC1-s is about 10-fold more effective than heterologous hC1-s in cleaving mC4, but mC1-s itself does not distinguish between human and murine C4, cleaving both equally well. Hence mC1-s does not exhibit the species specificity previously found for hC1-s, which shows a several hundred-fold preference for homologous human C4 over murine C4.
Asunto(s)
Complemento C1s/metabolismo , Animales , Sitios de Unión , Proteínas Sanguíneas/metabolismo , Complemento C4/metabolismo , Humanos , Técnicas In Vitro , Ratones , Especificidad de la Especie , Especificidad por SustratoRESUMEN
We have constructed cell surface-bound forms of murine C4b-binding protein (mC4BP) that allowed us to monitor the binding of mC4BP to C4b with relatively simple erythrocyte rosette assays. We used two types of surface-bound mC4BP: one in which segments of mC4BP were fused directly to a peptide containing the transmembrane and cytoplasmic domains of human complement receptor CR2 (BPR1-type); and a second in which the same segments were fused to a longer peptide containing the five membrane-proximal short consensus repeats (SCR) of CR2 as well as the transmembrane and cytoplasmic domains (BPR2-type). COS cells transfected with either construct carrying all six mC4BP SCR rosetted with C4b-bearing EAC14 cells but not with C4b-lacking EAC1 cells; and rosetting was inhibited by excess inactivated C4 but not inactivated C3. COS cells transfected with BPR2 constructs carrying only SCR 1-3 or 1-4 gave similar rosetting behavior. However, rosetting was not observed with BPR2 constructs carrying only SCR 1-2 or 2-6, or with BPR1 constructs carrying only SCR 1-2, 1-3, 1-4, or 2-6. Finally, we found that alteration of the AUG sequence 56 triplet codons upstream of the putative N-terminus of mature mC4-BP eliminates rosetting whereas alteration of a second AUG sequence 13 codons upstream has no effect on rosetting. These results indicate that 1) SCR 1-3 of mC4BP are necessary and adequate for binding to C4b, 2) steric effects close to the cell surface may interfere with binding, and 3) mC4BP has an extraordinarily long 56 amino acid residue signal peptide.
Asunto(s)
Proteínas Portadoras/química , Complemento C4b/metabolismo , Proteínas Inactivadoras de Complemento , Glicoproteínas , Precursores de Proteínas/química , Receptores de Complemento/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Relación Estructura-ActividadRESUMEN
CR2, a membrane glycoprotein, is one of a number of cell-surface proteins which bind activation and processing fragments of the complement system. CR2, which is found on normal B lymphocytes, follicular dendritic cells in lymphoid organs, and epithelial cells, interacts preferentially with C3dg, the terminal activation/processing fragment of the third complement component. Attachment of C3dg to CR2 brings complement activators, bearing covalently bound C3dg, into direct membrane contact with CR2-bearing cells. Epstein-Barr virus, a human herpesvirus, also binds to CR2 on B lymphocytes. Attachment of EBV is followed by infection. CR2 has been purified and the binding properties of its ligands analyzed. Monoclonal antibodies have been developed and used to probe the structural correlates of CR2 functions. CR2 has been molecularly cloned and its primary amino acid sequence deduced. These data indicate that it shares characteristic structural features with a number of other complement and non-complement cell membrane and plasma proteins. Several of the complement-associated proteins in this family possess regulatory functions; they are encoded by linked genes which have been localized to band q32 on chromosome 1. CR2 has been expressed in primate and rodent cells by transfection of cDNA in antigenically and functionally intact form. It has also been expressed in soluble form and its structure, electron microscopic appearance and binding characteristics analyzed in detail. The present state of knowledge of the structure and genetics of CR2 and current understanding of its biologic functions are summarized here.
Asunto(s)
Receptores de Complemento/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Fenómenos Químicos , Química , Complemento C3d/metabolismo , Células Dendríticas/metabolismo , Epitelio/metabolismo , Herpesvirus Humano 4/metabolismo , Humanos , Receptores de Complemento 3dRESUMEN
Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.
Asunto(s)
Proteínas Sanguíneas/fisiología , Complemento C4/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/genética , Clonación Molecular , Complemento C4/genética , ADN/genética , Hemólisis , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
Previously cloned and sequenced full-length cDNAs for murine C4 and the closely related sex-limited protein (Slp) have been placed into an eucaryotic expression vector. Transfer of these DNA constructs transiently into monkey COS cells or stably into mouse L cells results in the expression and secretion of hemolytically active mouse C4 and mature Slp. We estimate from hemolytic activities that COS and L cells secrete 0.04 and 3%, respectively, of the C4 level found in mouse plasma. Slp expression is consistently only 10-20% that of C4 although the identical expression system is used for both. Our results show subtle but reproducible cell-type-specific differences in C4 maturation; they also indicate that surprisingly large shifts in electrophoretic mobility on SDS-polyacrylamide gels are induced by a small number of amino acid substitutions. The expression of C4 from cDNAs of known sequence provides a starting point for studies of structure/function relationships in C4 employing site-specific mutagenesis and gene transfer.
Asunto(s)
Complemento C4/genética , Hemólisis/genética , Transfección/genética , Animales , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Complemento C4/análisis , Complemento C4/metabolismo , Ensayo de Actividad Hemolítica de Complemento , Haplorrinos , Técnicas In Vitro , Ratones , Plásmidos , Pruebas de PrecipitinaRESUMEN
Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.
Asunto(s)
Activación de Complemento , Complemento C3/metabolismo , Infecciones por Herpesviridae/fisiopatología , Herpesvirus Humano 4/fisiología , Animales , Complemento C3b/metabolismo , Complemento C4/metabolismo , Complemento C4b , Factor I de Complemento , Humanos , Técnicas In Vitro , Unión Proteica , Receptores de Complemento/metabolismo , Receptores de Complemento 3b , Serina Endopeptidasas/metabolismoRESUMEN
The EBV-producing B lymphoblastoid cell line B95-8 was found to efficiently activate the alternative C pathway whether assessed with Mg-EGTA-treated human serum or with mixtures of the purified proteins of the pathway (PAP). The ability of the cells to activate was markedly increased after stimulation of EBV replication by treatment of the cells with a phorbol ester, and decreased by treatment of the cells with a viral polymerase inhibitor. Alternative pathway activation was dependent on the presence of either properdin or EBV-immune IgG; the addition of either alone to the PAP led to the deposition of 200,000 C3 molecules/cell. The addition of both properdin and immune IgG to the PAP markedly increased C3 binding to a level of 800,000 molecules/cell. Several lines of evidence indicate that the major external glycoprotein of EBV, gp350, mediates alternative pathway activation by B95-8 cells. First, the ability to activate C positively correlated with gp350 expression on the surface of the EBV-producing cells and gp350- cells failed to activate; second, the anti-EBV antibody in immune human sera which enhanced activation specifically immunoprecipitated gp350 from membranes of B95-8 cells; third, a significant proportion of the C3 which became bound to the cells during activation was attached either to gp350 or to the anti-gp350 antibody found in immune human sera; and fourth, purified gp350, as well as EBV, efficiently activated the alternative pathway. These results indicate that gp350, an EBV envelope glycoprotein, is an efficient alternative pathway activator and its expression on cell membranes is associated with the ability to activate C.
Asunto(s)
Activación de Complemento , Vía Alternativa del Complemento , Herpesvirus Humano 4/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/fisiología , Reacciones Antígeno-Anticuerpo , Antígenos Virales/análisis , Callitrichinae , Línea Celular , Transformación Celular Viral/efectos de los fármacos , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Linfoma/inmunología , Linfoma/metabolismo , Linfoma/microbiología , Antígeno de Macrófago-1 , Properdina/fisiología , Receptores de Complemento/análisis , Acetato de Tetradecanoilforbol/farmacología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.