RESUMO
Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.
Assuntos
Anticorpos Monoclonais/genética , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Antígenos/imunologia , Sequência de Bases , Separação Celular , Primers do DNA/genética , Evolução Molecular Direcionada , Descoberta de Drogas , Citometria de Fluxo , Biblioteca Gênica , Células HEK293 , Humanos , Dados de Sequência Molecular , Ligação Proteica , Engenharia de ProteínasRESUMO
B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.
Assuntos
Linfócitos B/imunologia , Depleção Linfocítica , Linfócitos T Reguladores/imunologia , Tireoidite Autoimune/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/metabolismo , Tireoidite Autoimune/imunologia , Tireoidite Autoimune/patologiaRESUMO
B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have also long been considered central players in systemic lupus erythematosus. However, anti-CD20-mediated B cell depletion was not effective in two clinical lupus studies, whereas anti-B lymphocyte stimulator, which inhibits B cell survival, was effective. Others and we previously found that anti-CD20-based depletion was surprisingly ineffective in tissues of lupus-prone mice, but that persistent high doses eventually led to depletion and ameliorated lupus. Lupus patients might also have incomplete depletion, as suggested in several studies, and which could have led to therapeutic failure. In this study, we investigated the mechanism of resistance to Ab-mediated cellular depletion in murine lupus. B cells from lupus-prone mice were easily depleted when transferred into normal environments or in lupus-prone mice that lacked serum Ig. Serum from lupus-prone mice transferred depletion resistance, with the active component being IgG. Because depletion is FcγR-dependent, we assayed macrophages and neutrophils exposed to lupus mouse serum, showing that they are impaired in IgG-mediated phagocytosis. We conclude that depletion resistance is an acquired, reversible phagocytic defect depending on exposure to lupus serum IgG. These results have implications for optimizing and monitoring cellular depletion therapy.
Assuntos
Linfócitos B/imunologia , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Depleção Linfocítica , Fagocitose/imunologia , Animais , Anticorpos Monoclonais Murinos/farmacologia , Antígenos CD20/imunologia , Antirreumáticos/farmacologia , Separação Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoglobulina G/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/terapia , Depleção Linfocítica/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Camundongos Transgênicos , RituximabRESUMO
The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4(+) T cells expressing Foxp3 and CD25. On a per-cell basis, CD4(+)CD25(+) cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4(+) T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppressing Treg cells.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Subpopulações de Linfócitos B/imunologia , Depleção Linfocítica , Linfopenia/imunologia , Linfopenia/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/prevenção & controle , Subpopulações de Linfócitos B/patologia , Epitopos de Linfócito T/imunologia , Feminino , Inflamação/imunologia , Inflamação/patologia , Depleção Linfocítica/métodos , Linfopenia/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteoglicanas/administração & dosagem , Proteoglicanas/imunologia , Proteoglicanas/toxicidade , Índice de Gravidade de Doença , Linfócitos T Reguladores/patologiaRESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Doenças Autoimunes/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Imunossupressores/farmacologia , Mediadores da Inflamação/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Humanos , Imunossupressores/química , Imunossupressores/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/síntese química , Mediadores da Inflamação/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Pirróis/síntese química , Pirróis/uso terapêutico , RatosRESUMO
Inhibitors of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscape for patients with cancer. Clinical activity of anti-PD-(L)1 antibodies has resulted in increased median overall survival and durable responses in patients across selected tumor types. To date, 6 PD-1 and PD-L1, here collectively referred to as PD-(L)1, pathway inhibitors are approved by the US Food and Drug Administration for clinical use. The availability of multiple anti-PD-(L)1 antibodies provides treatment and dosing regimen choice for patients with cancer. Here, we describe the nonclinical characterization of dostarlimab (TSR-042), a humanized anti-PD-1 antibody, which binds with high affinity to human PD-1 and effectively inhibits its interaction with its ligands, PD-L1 and PD-L2. Dostarlimab enhanced effector T-cell functions, including cytokine production, in vitro. Since dostarlimab does not bind mouse PD-1, its single-agent antitumor activity was evaluated using humanized mouse models. In this model system, dostarlimab demonstrated antitumor activity as assessed by tumor growth inhibition, which was associated with increased infiltration of immune cells. Single-dose and 4-week repeat-dose toxicology studies in cynomolgus monkeys indicated that dostarlimab was well tolerated. In a clinical setting, based on data from the GARNET trial, dostarlimab (Jemperli) was approved for the treatment of adult patients with mismatch repair-deficient recurrent or advanced endometrial cancer that had progressed on or following prior treatment with a platinum-containing regimen. Taken together, these data demonstrate that dostarlimab is a potent anti-PD-1 receptor antagonist, with properties that support its continued clinical investigation in patients with cancer.
Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos , Neoplasias Experimentais , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Células CHO , Cricetulus , Humanos , Células Jurkat , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: A human Fcgamma-Fcepsilon fusion protein (GE2) designed to inhibit FcepsilonRI signaling by coaggregating FcepsilonRI with the inhibitory receptor FcgammaRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcgammaRIIB. OBJECTIVE: Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration. METHODS: The in vitro activity of mGE on mediator release and signaling pathways was characterized in IgE-sensitized bone marrow-derived mast cells (BMMCs). The in vivo activity of mGE was examined in mouse passive cutaneous and passive systemic anaphylaxis models, and the therapeutic activity of hGE2 was evaluated in Ascaris suum-sensitized cynomolgus monkeys. RESULTS: mGE inhibited release of histamine and cytokines by BMMCs from wild-type mice but not by BMMCs from FcgammaRIIB-deficient mice. In mice mGE blocked IgE-dependent anaphylaxis mediated by mast cells with sustained efficacy. In BMMCs mGE decreased spleen tyrosine kinase and extracellular signal-regulated kinases 1/2 phosphorylation and induced FcgammaRIIB phosphorylation and the subsequent recruitment of SH2 domain-containing inositol polyphosphate 5' phosphatase (SHIP) 1 and SH2 domain-containing protein tyrosine phosphatase (SHP) 1/2 phosphatases. When administered therapeutically, hGE2 protected sensitized monkeys from local anaphylaxis for 3 weeks. CONCLUSION: mGE-mediated inhibition of mast cell activation is associated with inhibitory signaling through FcgammaRIIB that results from activation of SHIP-1 and SHP-1/2 phosphatases.
Assuntos
Mastócitos/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Domínios de Homologia de src , Anafilaxia/prevenção & controle , Animais , Ascaris suum , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imunização , Imunoglobulina E/metabolismo , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusão/genética , Quinase Syk , Receptor 4 Toll-Like/metabolismoRESUMO
Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.
Assuntos
Asma/imunologia , Basófilos/imunologia , Mastócitos/imunologia , Pyroglyphidae/imunologia , Receptores de IgE/administração & dosagem , Receptores de IgG/administração & dosagem , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Asma/metabolismo , Asma/terapia , Basófilos/metabolismo , Liberação de Histamina , Humanos , Macaca fascicularis , Masculino , Mastócitos/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
BACKGROUND: B cell depletion by anti-CD20 antibody is used in desensitization protocols and for treatment of antibody-mediated rejection (AMR). However, little is known about the efficacy and the mechanism(s) of action. METHODS: A mouse model of HLA sensitization was used to study the effectiveness of anti-CD20 treatment on B cell depletion and anti-HLA antibody suppression. RESULTS: Immunization of C57BL/6 mice with skin grafts from a transgenic C57BL.Tg/HLA-A2.1 mouse resulted in robust production of anti-HLA IgM and IgG antibodies, and accelerated rejection of a secondary skin allograft (within 3 days) featured by intragraft IgG and C4d deposition. Both IgM and IgG alloantibodies are specific to HLA-A2 as well as to a panel of class I HLA, including A1, A3, A25, A26, A29, and A30. These alloantibodies were complement-dependently cytotoxic (CDC) against HLA-A2 expressing target cells. Administration of 2 doses of a mouse-anti-mouse CD20 monoclonal antibody significantly reduced the levels of anti-HLA IgG2a antibodies, suppressed serum CDC, and prolonged survival of the secondary skin allografts. Suppression of anti-HLA IgG antibodies was associated with significant depletion of B220(+)/CD5(-) B cells from the blood, the spleen and the bone marrow of the treated animals. CONCLUSION: Anti-CD20 treatment effectively depletes B220(+)/CD5(-) B cells, resulting in potent suppression of anti-HLA IgG and prolongation of skin graft survival. The data are in support for the use of anti-CD20 antibodies in highly-HLA sensitized patients undergoing desensitization and for the treatment of AMR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Formação de Anticorpos/efeitos dos fármacos , Antígenos CD20/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Citotoxicidade Imunológica , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Humanos , Imunização Passiva , Imunoglobulinas/sangue , Isoanticorpos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Pele/imunologiaRESUMO
Signaling through the CD40 receptor activates diverse molecular pathways in a variety of immune cell types. To study CD40 signaling complexes in B cells, we produced soluble CD40 cytoplasmic domain multimers that translocate across cell membranes and engage intracellular CD40 signaling pathways. As visualized by fluorescence microscopy, rapid transduction of recombinant Antennapedia-isoleucine zipper (Izip)-CD40 cytoplasmic domain fusion protein (Antp-CD40) occurred in both the DND39 B cell line and human tonsillar B cells. Upon cellular entry, Antp-CD40 activated NF-kappaB-dependent transcription, induced proteolytic processing of p100 to the p52/NF-kappaB2 subunit, and increased expression of CD80 and CD54 on the surface of B cells. Antp-CD40 transduction of B cells did not, however, activate detectable levels of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase and did not up-regulate CD95 expression. Analysis of Antp-CD40 complexes recovered from transduced B cells revealed that Antp-CD40 associated with endogenous TRAF3 and Ku proteins. Multimerization of Antp-CD40, or extensive clustering of transmembrane CD40, diminished the disruptive effect of the T254A mutation in the TRAF2/3 binding site of the CD40 cytoplasmic domain. Taken together, these results indicate that Antp-CD40 mimics some of the natural CD40 signaling pathways in B cells by assembling partially functional signaling intermediates that do not require plasma membrane localization. We present a novel approach for delivering pre-activated, soluble receptor cytoplasmic domains into cells and recovering intact signaling complexes for molecular analysis.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Antígenos CD40/química , Antígenos CD40/genética , Linhagem Celular , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF , TransfecçãoRESUMO
Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface. The unique capability of mammalian cells to present IgG in its fully post-translationally modified format also allows selection of antibodies for functional properties. One limitation of mammalian cell based systems, however, has been the smaller library size that can be presented compared to phage display approaches. Until recently, this has necessitated the use of libraries biased toward a particular antigen, such as libraries derived from immunized donors, to achieve success. An alternative approach has now been developed which recapitulates key aspects of the in vivo immune system through reproducing somatic hypermutation (SHM) in vitro. Libraries representing a naïve human B lymphocyte antibody repertoire are created by PCR amplification of the rearranged (D)J segments of heavy and light chain variable regions from human donors and incorporating the resulting sequence diversity into panels of human germline VH and VL genes. The resulting antibodies are presented as full length IgG on the surface of HEK293 cells. After isolation of antibodies binding to individual target antigens, subsequent affinity maturation using in vitro SHM is induced by expression of activation-induced cytidine deaminase (AID). Selection of antibodies from naïve fully human libraries using mammalian cell display coupled with in vitro SHM is an efficient methodology for the generation of high affinity human antibodies with excellent properties for drug development.
Assuntos
Anticorpos , Técnicas de Visualização da Superfície Celular , Hipermutação Somática de Imunoglobulina , Animais , Afinidade de Anticorpos , Citidina Desaminase , HumanosRESUMO
Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Humanos , Difração de Raios XRESUMO
OBJECTIVE: Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell-depleting agents, as well as attempts seeking diabetes prevention, are being considered. RESEARCH DESIGN AND METHODS: Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset. RESULTS: The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell-dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets. CONCLUSIONS: These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Antígenos CD20/metabolismo , Linfócitos B/efeitos dos fármacos , Diabetes Mellitus Tipo 1/metabolismo , Hipoglicemiantes/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Animais , Antígenos CD20/química , Autoanticorpos/análise , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Progressão da Doença , Feminino , Ilhotas Pancreáticas/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Estado Pré-Diabético/sangue , Estado Pré-Diabético/imunologia , Estado Pré-Diabético/metabolismo , RituximabRESUMO
Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.
Assuntos
Autoimunidade/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Tolerância Imunológica , Anemia/sangue , Anemia/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Complexo Antígeno-Anticorpo/metabolismo , Artrite/imunologia , Artrite/patologia , Autoantígenos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/mortalidade , Doenças Autoimunes/patologia , Autoimunidade/genética , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/farmacologia , Subpopulações de Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/transplante , Diferenciação Celular/genética , Movimento Celular/genética , Movimento Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Homeostase/imunologia , Rim/metabolismo , Rim/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quimera por Radiação/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T/citologiaRESUMO
B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50-80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.
Assuntos
Antígenos CD20/imunologia , Linfócitos B/imunologia , Glândula Tireoide/imunologia , Tireoidite Autoimune/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Tireoidite Autoimune/metabolismoRESUMO
The role played by B cells in cancer biology is complex and somewhat controversial. Previous studies using genetically engineered mice suggest that B cells may be immunosuppressive and inhibit tumor rejection. However, the effects of B-cell depletion employing an antibody in mice bearing solid tumors has not been tested owing to difficulties in making an effective antimouse CD20 antibody (similar to rituximab). Injection of a newly developed antimouse CD20 antibody was effective in depleting circulating B cells from blood and lymph nodes, although depletion was less complete in the spleen. B-cell depletion slowed the growth of new solid tumors (not expressing CD20) and retarded the growth of established tumors but did not induce tumor regression. However, when the antibody was combined with an active immunotherapy approach using an adenovirus vaccine expressing the human papilloma virus-E7 gene (Ad.E7) in mice bearing TC1 tumors (murine lung cancer cells expressing human papilloma virus-E7), we noted enhanced antitumor effects and increased numbers of tetramer+/CD8+ T cells within the spleens and activated CD8+ T cells within tumors. B-cell depletion using an anti-CD20 antibody was thus effective in retarding tumor growth in multiple solid tumor models and augmenting immunotherapy in a tumor vaccine model. These studies raise the possibility that B-cell depletion may be a useful adjunct in human immunotherapy trials.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Linfócitos B/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Adenoviridae/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Vetores Genéticos/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Cinética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/patologia , Baço/imunologiaRESUMO
B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood. In the proteoglycan-induced arthritis (PGIA) mouse model of RA, we reported that Ag-specific B cells have two important functions in the development of arthritis. PG-specific B cells are required as autoantibody-producing cells as well as Ag-specific APCs. Herein we report on the effects of anti-CD20 mAb B cell depletion therapy in PGIA. Mice were sensitized to PG and treated with anti-CD20 Ab at a time when PG-specific autoantibodies and T cell activation were evident but before acute arthritis. In mice treated with anti-CD20 mAb, development of arthritis was significantly reduced in comparison to control mAb-treated mice. B cell depletion reduced the PG-specific autoantibody response. Furthermore, there was a significant reduction in the PG-specific CD4(+) T cell recall response as well as significantly fewer PG-specific CD4(+) T cells producing IFN-gamma and IL-17, but not IL-4. The reduction in PG-specific T cells was confirmed by the inability of CD4(+) T cells from B cell-depleted mice to adoptively transfer disease into SCID mice. Overall, B cell depletion during PGIA significantly reduced disease and inhibited both autoreactive B cell and T cell function.
Assuntos
Antígenos CD20/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteoglicanas/farmacologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/imunologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Células Cultivadas , Feminino , Imunoterapia , CamundongosRESUMO
Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-gamma after herpes simplex virus (HSV) 2 infection. IFN-gamma acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa. Importantly, unlike other known Th1 defense mechanisms, this effector function did not require recognition of virally infected cells via MHC class II. Instead, recall Th1 response was elicited by MHC class II(+) antigen-presenting cells at the site of infection. Dendritic cells (DCs) were not required and only partially sufficient to induce a recall response from memory Th1 cells. Importantly, DCs and B cells together contributed to restimulating memory CD4 T cells to secrete IFN-gamma. In the absence of both DCs and B cells, immunized mice rapidly succumbed to HSV-2 infection and death. Thus, these results revealed a distinct mechanism by which memory Th1 cells mediate noncytolytic IFN-gamma-dependent antiviral protection after recognition of processed viral antigens by local DCs and B cells.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Herpesvirus Humano 2/imunologia , Memória Imunológica/imunologia , Células Th1/imunologia , Transferência Adotiva , Animais , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteína Ligante Fas/imunologia , Feminino , Herpesvirus Humano 2/fisiologia , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/imunologia , Perforina/genética , Perforina/imunologia , Vagina/citologia , Vagina/imunologia , Vagina/virologia , Replicação Viral , Receptor fas/imunologiaRESUMO
In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Linfócitos B/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/terapia , Depleção Linfocítica , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Antígenos CD20/genética , Autoanticorpos/sangue , Linfócitos B/imunologia , Contagem de Células , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Glomérulos Renais/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Transgênicos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcepsilon and IgG1-Fc (Fcgamma). Fcepsilon was found to be significantly less stable than Fcgamma under all pH and NaCl conditions tested. Additionally, the Cepsilon3Cepsilon4 domains of Fcepsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcepsilon and an Fcgamma-FcepsilonRIalpha fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcepsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 10(-12) m). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcepsilon unfolded, and interaction of Fcepsilon with the Fcgamma-FcepsilonRIalpha fusion protein was abrogated. The unusual pH sensitivity of Fcepsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.