RESUMO
Various types of structural variants have been observed in recombinant DNA - derived products. These isoforms include variations in post translational carbohydrate modifications where variations in site occupancy or unoccupied sites may occur. In addition, varying degrees of C-terminal processing and N-terminal substitutions have been observed. Isoforms may also be generated during processing and can include aggregated and/or chemically modified forms of the protein. Sophisticated analytical techniques exist for the identification and characterization of these structural variants. Several strategies have been used to isolate or enrich the isoform before molecular characterization. However, the effect these structural variations have on the biological activity of the product is less well understood. This may, in part, be due to the specificity and variability of the bioassay employed. This presentation describes the isolation and characterization of specific molecular isoforms for a monoclonal antibody product as well as an assessment of effects on biological activity.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas Recombinantes/química , Animais , Anticorpos Monoclonais/metabolismo , Indústria Farmacêutica/métodos , Eletroforese , Humanos , Espectrometria de Massas , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Stimulation by CD40 ligand (L) improves B-cell malignancy immunogenicity, and also induces proliferative signals. To avoid these tumorigenic effects, we studied an alternate way of tumor-cell stimulation by homologous to lymphotoxin, inducible expression, competing for GpD of herpesvirus, which binds to the herpesvirus entry mediator (HVEM), and is expressed on T-lymphocytes (LIGHT), the ligand for HVEM, a new member of the tumor necrosis factor (TNF)/TNF-receptor (-R) family. HVEM is constitutively expressed on the surface of tumor B cells. We focused our attention on mantle cell lymphoma, a subtype of B-cell malignancy of poor prognosis. Triggering by LIGHT, in contrast to CD40L stimulation, did not increase lymphoma proliferation nor decrease chemotherapy entrance. We observed an upregulation of the TNFR apoptosis-inducing ligand Fas, and in contrast to CD40L-induced protection, an enhancement of lymphoma sensitivity to Fas-induced apoptosis. LIGHT triggering increased lymphoma cell recognition in a mixed lymphocyte response. In conclusion, LIGHT-mediated triggering renders B-cell lymphomas more immunogenic and sensitive to apoptosis, without inducing proliferation. Since LIGHT triggering also enhances the functions of T-lymphocytes and dendritic cells, it could be a unique way to restore an efficient cancer control by its pleiotropic effects on immune effectors and tumor cells.
Assuntos
Apoptose/genética , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Receptores do Fator de Necrose Tumoral/genética , Receptores Virais/genética , Receptor fas/metabolismo , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/metabolismo , Adesão Celular/imunologia , Morte Celular/imunologia , Divisão Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Expressão Gênica , Humanos , Imunoterapia , Interleucina-2/metabolismo , Ligantes , Teste de Cultura Mista de Linfócitos , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/terapia , Membro 14 de Receptores do Fator de Necrose Tumoral , TransfecçãoRESUMO
Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in their heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. These antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking induced cell death in vitro as a link to differential CD4 cell depletion in vivo. We developed a simple flow cytometry procedure that measures apoptosis within intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), express external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA), within the intact and compromised lymphocyte populations. Treatment of freshly isolated PBMCs with keliximab resulted in the appearance of characteristic apoptotic condensed CD4 T cells that contained reactive oxygen species, were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In contrast, clenoliximab alone did not induce apoptosis. The use of multiparameter flow cytometry and Attractors to analyze subpopulations based on scatter properties and biochemical processes during apoptosis provides a sensitive assay in which to quantitate and characterize the induction of cell death. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, antibody-mediated apoptosis of these cells that is dependent on Fcgamma receptors.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Anexina A5/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo/estatística & dados numéricos , Fluoresceínas , Humanos , Técnicas In Vitro , Espécies Reativas de Oxigênio/metabolismo , Software , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.
Assuntos
Ligante de CD40/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Proteínas de Membrana/imunologia , Linfócitos T Citotóxicos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Ligante de CD40/genética , Diferenciação Celular , Linhagem Celular , Citocinas/biossíntese , Humanos , Imunoterapia , Técnicas In Vitro , Interferon gama/biossíntese , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Neoplasias/terapia , Pinocitose , Receptores do Fator de Necrose Tumoral/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral , Receptores Virais/imunologia , Transdução de Sinais , Transfecção , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genéticaRESUMO
Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The gamma4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcgamma receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t(1/2) of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitro and in vivo results demonstrate the potential of this IgG4-PE mAb for use in human trials.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Depleção Linfocítica , Pan troglodytes/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Artrite Reumatoide/terapia , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Clonagem Molecular , Genes de Imunoglobulinas , Humanos , Regiões Constantes de Imunoglobulina/genética , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Terapia de Imunossupressão/métodos , Macaca fascicularis , Masculino , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Desnaturação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de IgG/metabolismo , Relação Estrutura-AtividadeRESUMO
The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.
Assuntos
Regulação para Baixo/imunologia , Ativação Linfocitária , Proteínas de Membrana/biossíntese , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores Virais/antagonistas & inibidores , Receptores Virais/biossíntese , Simplexvirus/imunologia , Subpopulações de Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Separação Celular , Células Cultivadas , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/fisiologia , Microscopia Confocal , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral , Receptores Virais/genética , Receptores Virais/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/imunologiaRESUMO
The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.
Assuntos
Anticorpos Monoclonais/toxicidade , Antígenos CD4/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Células CHO , Candidíase/imunologia , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Feminino , Citometria de Fluxo , Humanos , Hipersensibilidade Tardia/imunologia , Sistema Imunitário/crescimento & desenvolvimento , Hibridização in Situ Fluorescente , Teste de Cultura Mista de Linfócitos , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Testes para Micronúcleos , Infecções por Pneumocystis/imunologia , Reprodução/efeitos dos fármacosRESUMO
This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD4/química , Antígenos CD4/imunologia , Imunoglobulina G/química , Sítios de Ligação de Anticorpos , Calorimetria/métodos , Varredura Diferencial de Calorimetria/métodos , Variação Genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Cinética , Substâncias Macromoleculares , Microquímica/métodos , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Ressonância de Plasmônio de Superfície/métodos , TermodinâmicaRESUMO
TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D) = 2 nm) and OPG having the weakest (K(D) = 400 nm). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.
Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Células CHO , Calorimetria , Cricetinae , Primers do DNA , Humanos , Glicoproteínas de Membrana/genética , Pichia/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Temperatura , Fator de Necrose Tumoral alfa/genéticaRESUMO
Clenoliximab and keliximab are monkey/human chimeric monoclonal antibodies (mAbs) of the immunoglobulin G4 (IgG4) and IgG1 isotypes, respectively, that recognize the same epitope on human CD4. The two mAbs possess identical idiotypes and exhibit equal affinities for CD4. Upon administration of these mAbs to mice that express a human CD4 transgene, but not mouse CD4 (HuCD4/Tg mice), clenoliximab and keliximab exhibited similar kinetics of binding to CD4, and induced the same degree of CD4 modulation from the cell surface, although only keliximab mediated CD4+ T-cell depletion. Epicutaneous sensitization and challenge of HuCD4/Tg mice with the hapten oxazolone resulted in a contact sensitivity response characterized by tissue swelling, and the presence of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the local tissue. Administration of a single 2-mg dose of either clenoliximab or keliximab to HuCD4/Tg mice prior to sensitization significantly reduced post-challenge tissue swelling, and levels of IFN-gamma and IL-4, indicating that CD4+ T-cell depletion is not required for anti-CD4 mAb-mediated inhibition of contact sensitivity. Administration of either mAb prior to challenge failed to inhibit the contact sensitivity response, indicating differential sensitivity of the afferent and efferent phases of the response to inhibition by CD4-specific mAbs. Collectively, these data indicate that CD4 functions as a positive regulatory molecule in the contact sensitivity response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Dermatite de Contato/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD4/metabolismo , Relação Dose-Resposta Imunológica , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Transgênicos , Oxazolona/imunologiaRESUMO
Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.
Assuntos
Capsídeo/administração & dosagem , Dependovirus/imunologia , Infecções por Parvoviridae/imunologia , Animais , Anticorpos/farmacologia , Anticorpos Antivirais/sangue , Antígenos CD4/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Capsídeo/biossíntese , Capsídeo/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos , Isotipos de Imunoglobulinas/sangue , Injeções Intramusculares , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/prevenção & controle , Proteínas Recombinantes/administração & dosagem , Fatores de TempoRESUMO
Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.
Assuntos
Anticorpos Monoclonais/fisiologia , Antígenos CD4/imunologia , Imunoglobulina G/metabolismo , Receptores Fc/fisiologia , Animais , Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Sítios de Ligação de Anticorpos , Antígenos CD4/metabolismo , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Humanos , Hibridomas , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Macaca fascicularis , Receptores Fc/metabolismoRESUMO
The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.
Assuntos
Adenoviridae/genética , Anticorpos Monoclonais/administração & dosagem , Antígenos CD4/genética , Antígenos CD4/imunologia , Vetores Genéticos/administração & dosagem , Pulmão/imunologia , Adenoviridae/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fibrose Cística/genética , Fibrose Cística/imunologia , Fibrose Cística/terapia , Feminino , Terapia Genética/métodos , Vetores Genéticos/imunologia , Humanos , Injeções Intraperitoneais , Intubação Intratraqueal , Pulmão/metabolismo , Ativação Linfocitária/genética , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.
Assuntos
Antineoplásicos/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/farmacologia , Células HT29/efeitos dos fármacos , Humanos , Ligantes , Teste de Cultura Mista de Linfócitos , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Ligação Proteica , Membro 14 de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Distribuição Tecidual , Fator de Transcrição AP-1/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
TR2 (TNFR-related 2) is a recently identified member of the TNFR family with homology to TNFRII. We have demonstrated previously that TR2 mRNA is expressed in resting and activated human T cells and that TR2-Ig partially inhibits an allogeneic mixed leukocyte proliferation response. We now characterize TR2 further by the use of specific mAbs. Flow-cytometry analysis using TR2 mAbs confirmed that resting PBL express high levels of cell surface TR2, and that TR2 is widely expressed on all freshly isolated lymphocyte subpopulations. However, stimulation of purified T cells with either PHA or PHA plus PMA resulted in decreased surface expression within 48 h of activation before returning to resting levels at 72 h. TR2 mAbs inhibited CD4+ T cell proliferation in response to stimulation by immobilized CD3 or CD3 plus CD28 mAbs. Assay of culture supernatants by ELISA showed inhibition of TNF-alpha, IFN-gamma, IL-2, and IL-4 production, which, for IL-2 and TNF-alpha was also confirmed by intracellular cytokine staining. Furthermore, expression of activation markers on CD4+ T cells, including CD25, CD30, CD69, CD71, and OX40 (CD134), was inhibited. TR2 mAbs inhibited proliferation in a three-way MLR, and a response to soluble recall Ag, tetanus toxoid. In conclusion, these results suggest that TR2 is involved in the activation cascade of T cell responses and TR2 mAbs prevent optimal T cell proliferation, cytokine production, and expression of activation markers.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Citocinas/antagonistas & inibidores , Ativação Linfocitária/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores Virais/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Citocinas/biossíntese , Epitopos de Linfócito T/imunologia , Inibidores do Crescimento/farmacologia , Humanos , Imunossupressores/farmacologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Membro 14 de Receptores do Fator de Necrose Tumoral , Simplexvirus/imunologia , Solubilidade , Linfócitos T/citologia , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
Recent findings that the multiple-action neurohumoral antagonist carvedilol inhibits the mitogenic effects of a broad variety of mitogens and produces marked protection against neointima formation after balloon angioplasty injury prompted further study into the molecular and biochemical mechanism of action. In the present study, the effects of carvedilol on mitogen-activated protein (MAP) kinase activity and cell cycle progression were evaluated. Carvedilol produced significant concentration-dependent inhibition of mitogen-induced MAP kinase activity in rat smooth muscle cells. Furthermore, when MAP kinase was purified from mitogen-stimulated cells by FPLC Mono Q chromatography, carvedilol produced direct enzyme inhibition. In the cell-free assay, carvedilol (10 microM) produced 50% inhibition of MAP kinase activity. Cell flow cytometry studies revealed that quiescent rat aortic smooth muscle cells showed 96% of the cell population in the G0/G1 phase of the cell cycle. The addition of serum (10%) increased the number of cells in S and G2/M phases 20% to 40%, respectively. Carvedilol (10 microM) significantly decreased (30-50%) the number of cells in S and G2/M phase. In addition, carvedilol significantly inhibited (>70%) serum-induced stimulation of the S phase-specific marker thymidine kinase. These data suggest that the antimitogenic actions of carvedilol on vascular smooth muscle may be in part due to the inhibition of MAP kinase activity and regulation of cell cycle progression.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Propanolaminas/farmacologia , Animais , Carvedilol , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Músculo Liso Vascular/enzimologia , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia , Timidina Quinase/metabolismoRESUMO
A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterized in vitro and in vivo in chimpanzees. This MAb binds human CD4 with Kd of 1.0 nM and was also able to bind to human IgG Fc receptors (Fc gamma R). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50 of about 10.0 ng/mL. Therefore, a chimpanzee in vivo model was used to characterize CE9.1, CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in an in vivo dose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1-3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+ CD8- CD4- cells. The CD4 receptor was totally undetectable on these lymphocytes for 1-2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+ lymphocytes, an equivalent number of CD3+CD8- T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+ T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Deleção Clonal , Testes de Fixação de Complemento , Reações Cruzadas , Humanos , Injeções Intravenosas , Teste de Cultura Mista de Linfócitos , Pan troglodytes , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Especificidade da EspécieRESUMO
The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22-36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4-1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation.
Assuntos
Cromossomos Humanos Par 1 , Ativação Linfocitária , Receptores do Fator de Necrose Tumoral/genética , Receptores Virais , Adulto , Sequência de Aminoácidos , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Masculino , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral , Distribuição TecidualRESUMO
A novel (TL1), a recently described (TL2) TNF-like, and three recently described TNF receptor-like (TR1, TR2, TR3) molecules were identified by searching a cDNA database. TL1 and TL2 are type-II membrane proteins. TR2 and TR3 are type-I membrane proteins whereas TR1 appears to be a secreted protein. TL1, TL2, TR2 and TR3 were expressed in hematopoietic cells, whereas TR1 was not. Northern blots hybridized with the cDNA probes revealed multiple forms of RNA as well as inducible expression of TL1, TL2, TR2 and TR3. TL2 and TR3, in particular, were highly induced in activated CD4+ T cells. Radiation hybrid mapping localized TR1 and TL2 to 8q24 and 3q26, respectively, which are not near any known superfamily members. TL1 was mapped to 9q32, near CD30L (9q33) and TR2 and TR3 mapped to the region of chromosome 1 that contains the TNFR-II, 4-1BB, OX40 and CD30 gene cluster at 1p36. Only TR3 in this cluster possesses a death domain. Southern blot analysis revealed the presence of TL and TR genes in different mammalian species. TL2, TR1, TR2 and TR3 were recently described by others as TRAIL/Apo-2L, OPG, HVEM and DR3/WSL-1/Apo-3/TRAMP/LARD, respectively.
Assuntos
Células Sanguíneas/metabolismo , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Animais , Células Sanguíneas/citologia , Southern Blotting , Bovinos , Linhagem Celular , Galinhas , Mapeamento Cromossômico , Cães , Drosophila , Sistema Hematopoético/citologia , Humanos , Células Jurkat , Ligantes , Linfócitos/citologia , Linfócitos/metabolismo , Linfoma , Camundongos , Dados de Sequência Molecular , RNA , Coelhos , Ratos , Receptores do Fator de Necrose Tumoral/genética , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.