RESUMEN
While conventional chimeric antigen-receptor (CAR)-T therapies have shown remarkable clinical activity in some settings, they can induce severe toxicities and are rarely curative. To address these challenges, we developed a controllable cell therapy where synthetic D-domain-containing proteins (soluble protein antigen-receptor X-linker [SparX]) bind one or more tumor antigens and mark those cells for elimination by genetically modified T cells (antigen-receptor complex [ARC]-T). The chimeric antigen receptor was engineered with a D-domain that specifically binds to the SparX protein via a unique TAG, derived from human alpha-fetoprotein. The interaction is mediated through an epitope on the TAG that is occluded in the native alpha-fetoprotein molecule. In vitro and in vivo data demonstrate that the activation and cytolytic activity of ARC-T cells is dependent on the dose of SparX protein and only occurs when ARC-T cells are engaged with SparX proteins bound to antigen-positive cells. ARC-T cell specificity was also redirected in vivo by changing SparX proteins that recognized different tumor antigens to combat inherent or acquired tumor heterogeneity. The ARC-SparX platform is designed to expand patient and physician access to cell therapy by controlling potential toxicities through SparX dosing regimens and enhancing tumor elimination through sequential or simultaneous administration of SparX proteins engineered to bind different tumor antigens.
Asunto(s)
Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Animales , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Inmunoterapia Adoptiva/métodos , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Neoplasias/terapia , Neoplasias/inmunología , Neoplasias/metabolismo , Unión ProteicaRESUMEN
Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Anticuerpos de Cadena Única , Animales , Antígeno de Maduración de Linfocitos B/metabolismo , Humanos , Inmunoterapia Adoptiva , Ratones , Mieloma Múltiple/patología , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos de Cadena Única/genética , Linfocitos TRESUMEN
Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoterapia Adoptiva/métodos , Subunidad alfa del Receptor de Interleucina-3/inmunología , Dominios Proteicos/inmunología , Proteínas/uso terapéutico , Receptores Quiméricos de Antígenos/uso terapéutico , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Antineoplásicos/inmunología , Epítopos de Linfocito T/inmunología , Células HEK293 , Humanos , Células K562 , Leucemia Mieloide Aguda/terapia , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Unión Proteica/inmunología , Proteínas/inmunología , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Cadena Única/inmunología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Neoplasias/terapia , Péptidos/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Secuencia de Aminoácidos , Angiopoyetina 2/química , Angiopoyetina 2/genética , Angiopoyetina 2/inmunología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Proliferación Celular/efectos de los fármacos , Cetuximab , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Neovascularización Patológica , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal , Trastuzumab , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.
Asunto(s)
Angiopoyetina 2/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de Necrosis Tumoral alfa/inmunología , Adalimumab , Angiopoyetina 2/genética , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/terapia , Línea Celular , Modelos Animales de Enfermedad , Humanos , Inflamación/terapia , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Resultado del TratamientoRESUMEN
For the first time in more than 50 years, the US Food and Drug Administration has approved a drug specifically for the treatment of systemic lupus erythematosus (SLE). This drug, belimumab (Benlysta), is a human monoclonal antibody that neutralizes the B-cell survival factor, B-lymphocyte stimulator (BLyS). The approval of belimumab combined a pioneering approach to genomics-based gene discovery, an astute appreciation of translational medicine, a disciplined clinical strategy, a willingness to take calculated risks, a devoted cadre of patients and physicians and a healthy dose of serendipity. Collectively, these efforts have provided a model for the development of a new generation of drugs to treat the broad manifestations of SLE. However, as a substantial percentage of SLE patients do not respond to belimumab, further research is needed to better characterize the pathogenetic mechanisms of SLE, identify additional therapeutic targets, and develop effective and nontoxic novel agents against these targets.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Factor Activador de Células B/inmunología , Aprobación de Drogas , Lupus Eritematoso Sistémico/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados , Factor Activador de Células B/antagonistas & inhibidores , Factor Activador de Células B/genética , Linfocitos B/inmunología , Linfocitos B/patología , Ensayos Clínicos Controlados como Asunto , Resistencia a Medicamentos/genética , Humanos , Ratones , Resultado del Tratamiento , Estados Unidos , United States Food and Drug AdministrationRESUMEN
We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.
Asunto(s)
Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas/inmunología , Transducción de Señal/inmunología , Animales , Atrofia/genética , Atrofia/inmunología , Atrofia/patología , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Centro Germinal/patología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/inmunología , Inmunosupresores/farmacología , Ratones , Ratones Noqueados , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
OBJECTIVE: To assess the effects of B lymphocyte depletion on serum B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) levels in patients with rheumatoid arthritis (RA), and to assess the relationship of serum BLyS levels with peripheral blood B cell depletion, levels of autoantibodies and antimicrobial antibodies, the return of peripheral blood B cells, and clinical relapse. METHODS: Fifteen patients with active RA underwent rituximab-based B cell depletion therapy (BCDT). Disease activity was assessed clinically, peripheral blood CD19+ B cell counts were determined by flow cytometry, and serum levels of BLyS, IgM, IgA, and IgG rheumatoid factors (RFs), anti-cyclic citrullinated peptide, and antimicrobial antibodies were assessed using enzyme-linked immunosorbent assays. RESULTS: Peripheral blood B cell depletion was achieved in all 15 patients, and an American College of Rheumatology 20% response was achieved in 13 patients. Following clinical relapse, 7 patients underwent at least 1 additional cycle of BCDT. In every case, serum BLyS levels markedly rose post-BCDT and remained elevated for at least 1-2 months. Serum levels of RF, but not those of anti-tetanus toxoid or anti-pneumococcal polysaccharide antibodies, fell significantly. A decline in serum BLyS levels was associated with the reemergence of B cells in peripheral blood, which, in turn, antedated clinical relapse by variable periods of time. The patterns of B cell depletion, serum BLyS and antibody levels, and clinical relapse for each BCDT cycle were remarkably similar in re-treated patients. CONCLUSION: Rituximab-based BCDT leads to marked increases in serum BLyS levels. This may contribute significantly to the survival and/or regeneration of B cell populations capable of triggering clinical relapse.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos/sangre , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Depleción Linfocítica , Proteínas de la Membrana/sangre , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Anticuerpos Monoclonales de Origen Murino , Antígenos CD19/análisis , Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Factor Activador de Células B , Linfocitos B/inmunología , Femenino , Humanos , Depleción Linfocítica/métodos , Masculino , Persona de Mediana Edad , Recurrencia , Factor Reumatoide/sangre , Rituximab , Factor de Necrosis Tumoral alfaRESUMEN
Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and DeltaBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and DeltaBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and DeltaBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and DeltaBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.
Asunto(s)
Factor Activador de Células B/sangre , Factor Activador de Células B/genética , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/genética , ARN Mensajero/genética , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Factor Activador de Células B/inmunología , Humanos , Inmunoglobulinas/sangre , Lupus Eritematoso Sistémico/inmunología , Pacientes Ambulatorios , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/sangre , Isoformas de Proteínas/inmunologíaRESUMEN
B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.
Asunto(s)
Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/química , Secuencias de Aminoácidos , Factor Activador de Células B , Bioensayo , Secuencia Conservada , Disulfuros/química , Polarización de Fluorescencia , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Péptidos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptores del Factor de Necrosis Tumoral/fisiología , Rutenio/química , Proteína Activadora Transmembrana y Interactiva del CAML , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To assess the overexpression of B lymphocyte stimulator (BLyS) over time in patients with systemic lupus erythematosus (SLE). METHODS: Sixty-eight SLE patients were followed up longitudinally for a median 369 days. At each physician encounter, disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index, and blood was collected for determination of the serum BLyS level, blood BLyS messenger RNA (mRNA) level, and cell surface BLyS expression. Twenty normal control subjects underwent similar laboratory evaluations. RESULTS: In contrast to the uniformly normal serum BLyS and blood BLyS mRNA phenotypes in control subjects, SLE patients displayed marked heterogeneity, with 50% and 61% of patients manifesting persistently or intermittently elevated serum BLyS and blood BLyS mRNA phenotypes, respectively. Surface BLyS expression by SLE peripheral blood mononuclear cells was also often increased. Treatment of patients who had elevated serum BLyS levels with intensive courses of high-dose corticosteroids resulted in marked reductions in serum BLyS levels, and tapering of the corticosteroid dosage often resulted in increases in serum BLyS levels. Serum BLyS levels generally correlated with anti-double-stranded DNA (anti-dsDNA) titers (in those with detectable anti-dsDNA titers), but changes in serum BLyS levels did not correlate with changes in disease activity in individual patients. Serum BLyS phenotype did not associate with specific organ system involvement. CONCLUSION: Dysregulation of BLyS over extended periods of time is common in patients with SLE. Neutralization of BLyS activity with an appropriate BLyS antagonist may be therapeutically beneficial.
Asunto(s)
Linfocitos B/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/fisiopatología , Proteínas de la Membrana/genética , Factor de Necrosis Tumoral alfa/genética , Corticoesteroides/uso terapéutico , Adulto , Anciano , Antígenos de Superficie/genética , Autoanticuerpos/sangre , Factor Activador de Células B , ADN/inmunología , Femenino , Expresión Génica/inmunología , Humanos , Estudios Longitudinales , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , Proteínas de la Membrana/sangre , Persona de Mediana Edad , ARN Mensajero/sangreRESUMEN
OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Linfocitos B/metabolismo , Proteínas de la Membrana , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Receptor del Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Intravenosas , Leucocitos Mononucleares/efectos de los fármacos , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Macaca fascicularis , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Receptores del Factor de Necrosis Tumoral/inmunología , Bazo/citología , Bazo/efectos de los fármacos , Proteína Activadora Transmembrana y Interactiva del CAMLRESUMEN
B lymphocyte stimulator (BLyS protein) is a member of the human TNF family of ligands. BLyS induces B-lymphocyte proliferation and Ig secretion in vitro and in vivo. These qualities suggest that it may be useful as a therapeutic in the treatment of immunodeficiencies characterized by low or absent serum immunoglobulin, such as common variable immunodeficiency (CVID). CVID is characterized by the inability to generate adequate serum Ig despite normal or slightly depressed peripheral B, T, and myeloid cell populations. We tested the ability of BLyS to stimulate B lymphocytes obtained from CVID patients. Among five patients studied, 60% (three of five) produced normal quantities of IgM when cultured in the presence of BLyS. B-cell proliferation among patients was comparable, with 60% (three of five) responding to BLyS stimulation. These results suggest that BLyS induces proliferative and Ig-secretory responses in B lymphocytes isolated from some CVID patients and lend support to its potential use in therapy of this disorder.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/inmunología , Inmunodeficiencia Variable Común/inmunología , Inmunoglobulinas/inmunología , Proteínas de la Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Factor Activador de Células B , Receptor del Factor Activador de Células B , Linfocitos B/metabolismo , Baculoviridae/genética , División Celular/efectos de los fármacos , División Celular/inmunología , Inmunodeficiencia Variable Común/terapia , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Isotipos de Inmunoglobulinas , Inmunoglobulinas/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To determine whether synovial fluid (SF) levels and cell-surface expression of B lymphocyte stimulator (BLyS) protein and SF levels of APRIL are elevated in patients with inflammatory arthritis (IA). METHODS: Same-day blood and SF samples from 89 patients with 103 knee effusions (81 knees with IA and 22 with noninflammatory arthritis [NIA]) were evaluated for BLyS protein and APRIL levels by enzyme-linked immunosorbent assay. Blood and SF mononuclear cells were double-stained for surface BLyS protein and surface CD14 (monocyte marker) and were analyzed by flow cytometry. Complete blood cell counts and SF nucleated cell counts were performed by the clinical hematology laboratory. RESULTS: BLyS protein levels were higher in SF than in corresponding serum samples from both IA and NIA patients. SF BLyS protein levels, but not surface expression of BLyS protein, were disproportionately elevated in IA patients. APRIL levels were higher in SF than in corresponding serum samples from most IA patients but not NIA patients. SF BLyS protein and APRIL levels correlated with each other, and each correlated with SF monocyte, lymphocyte, neutrophil, and total nucleated cell counts. Although SF and serum BLyS protein levels correlated with each other, SF and serum APRIL levels did not, suggesting that SF BLyS protein levels are more dependent upon systemic factors than are SF APRIL levels. Moreover, in 8 patients who underwent sequential arthrocenteses, changes in SF BLyS protein levels did not immutably parallel changes in SF APRIL levels, indicating their differential regulation. CONCLUSION: BLyS protein and APRIL are locally produced in inflamed joints. Their respective SF levels are differentially regulated, suggesting that they serve different functions. Together, their local production may foster survival and/or expansion of B cells that produce pathogenic autoantibodies and/or promote local T cell activation and consequent joint destruction.
Asunto(s)
Artritis Reumatoide/metabolismo , Articulación de la Rodilla/metabolismo , Proteínas de la Membrana/metabolismo , Neuropéptidos/metabolismo , Proteínas Nucleares/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Factor Activador de Células B , Recuento de Células , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Receptores de Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/metabolismo , Líquido Sinovial/citología , Líquido Sinovial/metabolismoRESUMEN
BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers.
Asunto(s)
Linfocitos B/inmunología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/fisiología , Enfermedades Reumáticas/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Animales , Artritis Psoriásica/sangre , Artritis Psoriásica/inmunología , Artritis Reactiva/sangre , Artritis Reactiva/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Receptor del Factor Activador de Células B , Linfocitos B/metabolismo , Línea Celular , Células Cultivadas , Femenino , Humanos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/genética , Proteínas de la Membrana/sangre , Proteínas de la Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Polimiositis/sangre , Polimiositis/inmunología , Receptores del Factor de Necrosis Tumoral/sangre , Receptores del Factor de Necrosis Tumoral/aislamiento & purificación , Enfermedades Reumáticas/sangre , Espondilitis Anquilosante/sangre , Espondilitis Anquilosante/inmunología , Transfección , Células Tumorales Cultivadas , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/aislamiento & purificaciónRESUMEN
B Lymphocyte Stimulator (BLyS protein) is a member of the tumor necrosis factor (TNF) family of ligands and functions as an essential in vivo regulator of B lymphocyte homeostasis. As such, changes in systemic BlyS protein expression caused by disruption of the gene encoding BLyS or administration of neutralizing soluble receptors have resulted in profound losses in mature B cell numbers and impaired humoral immunity. A similar phenotype has been observed in A/WySnJ mice that express a truncated BLyS receptor and are thus defective in BLyS signal transduction. In contrast, overexpression of BLyS protein in BLyS-transgenic mice results in B cell hyperplasia, hypergammaglobulinemia, and development of autoimmune-like disease. The ability of BLyS to regulate both the size and repertoire of the peripheral B cell compartment raises the possibility that BLyS and antagonists thereof may form the basis of a therapeutic trichotomy. As an agonist, BLyS protein may enhance humoral immunity in congenital or acquired immunodeficiencies such as those resulting from viral infection or cancer therapy. BLyS-specific antagonists (antibodies or soluble receptors) that inhibit the biological activity of BLyS may be effective therapies for those autoimmune diseases characterized by polyclonal hypergammaglobulinemia and elevated autoantibody titers. Finally, the specificity of BLyS for B-lineage cells raises the possibility that BLyS may be used as a targeting vehicle for delivery of a cytotoxic or cytolytic signal to neoplastic B-lineage cells expressing one or more of the three known BLyS receptors. This review discusses the therapeutic potential of BLyS in the context of BLyS structure, function and receptor specificity.
Asunto(s)
Linfocitos B/inmunología , Enfermedades Hematológicas/inmunología , Proteínas de la Membrana/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Formación de Anticuerpos , Factor Activador de Células B , Receptor del Factor Activador de Células B , Linfocitos B/efectos de los fármacos , Enfermedades Hematológicas/tratamiento farmacológico , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/uso terapéutico , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/uso terapéuticoRESUMEN
To assess the helper T cell dependence of B lymphocyte stimulator (BLyS) protein-driven autoantibody production in vivo, serum levels of BLyS protein, total IgG, and anti-IgG anti-phospholipid (aPhL) autoantibodies from HIV-infected patients (n = 105) with varying degrees of CD4+ cell depletion and healthy control donors at low risk for HIV (n = 64) were determined. Peripheral blood mononuclear cells from these subjects were stained for surface expression of BLyS protein. Monocyte surface expression and serum levels of BLyS protein were increased in HIV-infected patients as were serum total IgG and IgG aPhL autoantibody levels. No associations were detected between increased serum BLyS protein levels and patient age, sex, disease duration, history of opportunistic infection or malignancy, or serum total IgG levels. However, serum levels of IgG aPhL autoantibodies were greater in patients with high serum BLyS protein levels than in those with normal serum BLyS protein levels. Importantly, this association between serum levels of BLyS protein and IgG aPhL was appreciated only in patients who were not severely CD4+ cell-depleted and not in patients who were severely CD4+ cell-depleted (peripheral blood CD4+ cell counts Asunto(s)
Linfocitos T CD4-Positivos/inmunología
, Infecciones por VIH/inmunología
, VIH-1
, VIH-2
, Leucocitos Mononucleares/inmunología
, Proteínas de la Membrana/análisis
, Factor de Necrosis Tumoral alfa/análisis
, Adulto
, Anciano
, Autoanticuerpos/sangre
, Factor Activador de Células B
, Recuento de Linfocito CD4
, Estudios de Cohortes
, Femenino
, Infecciones por VIH/sangre
, Humanos
, Inmunoglobulina G/sangre
, Masculino
, Proteínas de la Membrana/sangre
, Persona de Mediana Edad
, Fosfolípidos/inmunología
RESUMEN
Immunoglobulin (Ig) class-switch DNA recombination (CSR) is thought to be highly dependent upon engagement of CD40 on B cells by CD40 ligand on T cells. We show here that dendritic cells up-regulate BLyS and APRIL upon exposure to interferon-alpha, interferon-gamma or CD40 ligand. In the presence of interleukin 10 (IL-10) or transforming growth factor-beta, BLyS and APRIL induce CSR from C(mu) to C(gamma) and/or C(alpha) genes in B cells, whereas CSR to C(epsilon) requires IL-4. Secretion of class-switched antibodies requires additional stimulation by B cell antigen receptor engagement and IL-15. By eliciting CD40-independent Ig class switching and plasmacytoid differentiation, BLyS and APRIL critically link the innate and adaptive immune responses.
Asunto(s)
Antígenos CD40/fisiología , Células Dendríticas/fisiología , Cambio de Clase de Inmunoglobulina/genética , Proteínas de la Membrana/fisiología , Neuropéptidos/fisiología , Proteínas Nucleares/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Factor Activador de Células B , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Recombinación Genética , Regulación hacia ArribaRESUMEN
These studies characterize BLyS responsiveness and receptor expression among transitional and mature peripheral B cells. The results show a maturation-associated increase in BLyS binding capacity that reflects differential expression patterns of the three BLyS receptors. Accordingly, BLyS administration enlarges only late transitional and mature peripheral B (MB) cell compartments. Furthermore, bromodeoxyuridine labeling and cell cycle analyses show these effects are mediated through enhanced proportional survival of cells traversing the T2, T3, and MB cell stages, rather than by causing proliferation or slowing transit within these subsets. Despite similar effects on survival, BLyS up-regulates the antiapoptotic genes A1 and bcl-x(L) in MB cells but not immature B cells. Together, these findings show that, while BLyS influences B cell survival in several peripheral differentiation subsets, the downstream mediators differ, thus providing the first direct evidence for an established B lineage survival system whose intermediates change as B cells mature.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Proteínas de la Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Adyuvantes Inmunológicos/fisiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Factor Activador de Células B , Subgrupos de Linfocitos B/metabolismo , Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Supervivencia Celular/inmunología , Ratones , Antígenos de Histocompatibilidad Menor , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores del Factor de Necrosis Tumoral/biosíntesis , Regulación hacia Arriba/inmunología , Proteína bcl-XRESUMEN
OBJECTIVE: B Lymphocyte Stimulator (BLyS) protein and its receptor are new members of the tumor necrosis factor family, with specific effects exclusively on B cells. We have studied the tumor cell expression of the BLyS-Receptor (BLyS-R) and the serum BLyS protein levels in patients with different types of non-Hodgkin's lymphomas (NHL). METHODS: BLyS-R expression was assessed by flow cytometry on B cells from 43 NHL patients and 10 normal donors. BLyS protein serum levels were analyzed by ELISA. RESULTS: All B cells, tumor and normal, expressed BLyS-R. The mean fluorescence intensity (MFI +/- SD) of BLyS-R on normal B cells was 25.2 +/- 2.3 arbitrary units, while follicular NHL and chronic lymphocytic leukemia (CLL) exhibited significantly lower expression of the BLyS-R (17.7 +/- 3.1; 15.5 +/- 3.9, respectively, p < 0.0001 for both); other lymphoma subtypes expressed levels comparable to normal B cells (diffuse large cell, 24.8 +/- 4.3; mantle cell, 20 +/- 4.7; marginal zone, 20.7 +/- 3.7). BLyS protein serum levels were analyzed in 15 normal donors and 17 patients with follicular NHL. Levels of BLyS protein were, on average, threefold higher in patients with follicular lymphoma compared to normal donors (mean +/- SD; 13.4 +/- 5.6 ng/mL vs 4.6 +/- 0.7 ng/mL; p < 0.0001). BLyS protein alone was unable to stimulate proliferation in cultures of follicular lymphoma B cells or normal B cells. CONCLUSION: The specificity of the expression of BLyS-R by B-cell lymphomas opens new opportunities for the treatment of these cancers by targeting this ligand-receptor pair.