RESUMEN
Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Endocitosis/inmunología , Células Endoteliales/inmunología , Infecciones por VIH/inmunología , Receptores de IgG/metabolismo , Animales , Capilares/citología , Capilares/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/virología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Células HEK293 , VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/patología , Infecciones por VIH/virología , Voluntarios Sanos , Humanos , Hígado/irrigación sanguínea , Hígado/inmunología , Lisosomas/metabolismo , Lisosomas/virología , Masculino , Ratones , Ratones Noqueados , Cultivo Primario de Células , Receptores de IgG/genéticaRESUMEN
Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. Obexelimab is a non-depleting anti-human CD19 mAb with an Fc region engineered to have high affinity for human FcγRIIb, thereby co-engaging BCR and FcγRIIb. To assess its ability to suppress B cell activation in vivo, we generated non-autoimmune-prone C57BL/6 (B6) and SLE-prone NZM 2328 (NZM) mice in which the human FcγRIIb extracellular domain was knocked into the mouse Fcgr2b locus (B6.hRIIb and NZM.hRIIb mice, respectively, the latter retaining features of SLE). XENP8206, a mAb which bears the same FcγRIIb-enhanced human Fc domain as does obexelimab but which recognizes murine CD19 rather than human CD19, inhibited in vitro BCR-triggered activation of B cells from both B6.hRIIb and NZM.hRIIb mice. Following administration of XENP8206 to B6.hRIIb or NZM.hRIIb mice, B cell numbers in the spleen and lymph nodes remained stable but became hyporesponsive to BCR-triggered activation for at least 14 days. These findings demonstrate proof-of-principle that pharmacologic co-engagement of BCR and human FcγRIIb inhibits B cell activation in non-autoimmune and SLE-prone hosts while preserving B cell numbers. These observations lay a strong foundation for clinical trials in human SLE with agents that co-engage BCR and FcγRIIb. Moreover, B6.hRIIb and NZM.hRIIb should serve as powerful in vivo models in the elucidation of the cellular and molecular underpinnings of the changes induced by BCR/FcγRIIb co-engagement.
RESUMEN
Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigenâ¯×â¯CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Monoclonales , Ingeniería de Proteínas/métodos , Antígenos de Neoplasias/inmunología , Complejo CD3/inmunología , HumanosRESUMEN
OBJECTIVE: Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. METHODS: We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti-citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. RESULTS: B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. CONCLUSION: Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Antígenos CD19/inmunología , Artritis Reumatoide/patología , Linfocitos B/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/efectos de los fármacos , Receptores de IgG/efectos de los fármacos , Animales , Anticuerpos Antiidiotipos/metabolismo , Antígenos CD19/metabolismo , Artritis Reumatoide/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Antígeno B7-2/metabolismo , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Femenino , Xenoinjertos , Humanos , Leucocitos Mononucleares/patología , Ratones , Ratones SCID , Péptidos Cíclicos/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/metabolismoRESUMEN
The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.
Asunto(s)
Artritis Experimental/terapia , Artritis Reumatoide/terapia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Rechazo de Injerto/terapia , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoconjugados/metabolismo , Unión Proteica/efectos de los fármacos , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Abatacept , Animales , Afinidad de Anticuerpos , Formación de Anticuerpos/efectos de los fármacos , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Células Cultivadas , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/farmacología , Terapia de Inmunosupresión , Trasplante de Riñón , Activación de Linfocitos/efectos de los fármacos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos DBA , Mutación/genética , Unión Proteica/inmunología , Ingeniería de Proteínas , Receptores Fc/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Relación Estructura-ActividadRESUMEN
Mast cells and basophils play a central role in allergy, asthma, and anaphylaxis, as well as in non-allergic inflammatory, neurological and autoimmune diseases. Allergen-mediated cross-linking of IgE bound to FcεRI leads to cellular activation, and the low-affinity Fc receptor FcγRIIb is a key inhibitor of subsequent degranulation. FcγRIIb, when coengaged with FcεRI via allergen bound to IgE, stimulates ITIM domain-mediated inhibitory signaling that efficiently suppresses mast cell and basophil activation. To assess the therapeutic potential of directed coengagement of FcεRI and FcγRIIb in the absence of FcεRI crosslinking, we developed a fusion protein comprising the coupled Fc domains of murine IgE and human IgG1. As a key functional component of this tandem Fcε-Fcγ biologic, we engineered its IgG1 Fc domain to bind to human FcγRIIb with 100-fold enhanced affinity relative to native IgG1 Fc. Using mast cells from mice transgenic for human FcγRIIb, we show that this tandem Fc binds with high affinity to murine FcεRI and human FcγRIIb on mast cells, triggers phosphorylation of FcγRIIb, and inhibits FcεRI-dependent calcium mobilization. Control tandem Fc biologics containing a native IgG1 Fc domain or lacking binding to Fcγ receptors were markedly less active, demonstrating that the affinity-optimized tandem Fc can inhibit degranulation through stimulation of FcγRIIb signaling as well as through competition with allergen-IgE immune complex for FcεRI binding. We propose that in the context of a fully human tandem Fc biologic, high-affinity coengagement of FcεRI and FcγRIIb has potential as a novel therapy for allergy and other mast cell and basophil-mediated pathologies.
Asunto(s)
Degranulación de la Célula , Inmunoglobulina E/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Mastocitos/fisiología , Receptores de IgG/inmunología , Animales , Calcio/metabolismo , Diferenciación Celular , Humanos , Mastocitos/citología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Fosforilación , Proteínas Recombinantes/inmunologíaRESUMEN
HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Antígenos CD/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Mieloma Múltiple/terapia , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Degranulación de la Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Sinergismo Farmacológico , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Células Asesinas Naturales/inmunología , Lenalidomida , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Depleción Linfocítica , Macaca fascicularis , Ratones , Ratones SCID , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Talidomida/administración & dosificación , Talidomida/análogos & derivados , Talidomida/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Sequestration of IgE to prevent its binding to high-affinity IgE receptor FcεRI on basophils and mast cells is an effective therapy for allergic asthma. IgE production requires differentiation of activated IgE(+) B cells into plasma cells upon allergen sensitization. B-cell receptor signaling is suppressed by the inhibitory IgG Fc receptor FcγRIIb; therefore, we reasoned that a therapeutic antibody that coengages FcγRIIb and IgE B-cell receptor would not only sequester IgE but also suppress its production by blocking IgE(+) B-cell activation and differentiation to IgE-secreting plasma cells. OBJECTIVE: To explore the effects of IgE sequestration versus IgE suppression by comparing omalizumab to FcγRIIb-optimized anti-IgE antibodies in humanized mouse models of immunoglobulin production. METHODS: By using a murine anti-IgE antibody as a template, we humanized, increased IgE binding, and modified its Fc domain to increase affinity for FcγRIIb. We next compared effects of this antibody (XmAb7195) versus omalizumab on the secretion of IgE and other isotypes in human PBMC cultures and in PBMC-engrafted severe combined immunodeficiency mice. RESULTS: Relative to omalizumab, XmAb7195 has a 5-fold higher affinity for human IgE and more than 400-fold higher affinity for FcγRIIb. In addition to sequestering soluble IgE, XmAb7195 inhibited plasma cell differentiation and consequent human IgE production through coengagement of IgE B-cell receptor with FcγRIIb. In PBMC-engrafted mice, XmAb7195 reduced total human IgE (but not IgG or IgM) levels by up to 40-fold relative to omalizumab. CONCLUSION: XmAb7195 acts by IgE sequestration coupled with an FcγRIIb-mediated inhibitory mechanism to suppress the formation of IgE-secreting plasma cells and reduce both free and total IgE levels.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de IgE/antagonistas & inhibidores , Receptores de IgG/antagonistas & inhibidores , Animales , Antialérgicos/farmacología , Anticuerpos Antiidiotipos/sangre , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/genética , Afinidad de Anticuerpos/inmunología , Humanos , Inmunoglobulina E/metabolismo , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/trasplante , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Omalizumab , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgE/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMEN
Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens, and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Complejo CD3/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/metabolismo , Complejo CD3/genética , Complejo CD3/metabolismo , Dimerización , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Modelos Moleculares , Ingeniería de Proteínas/métodos , Receptores de IgG/genética , Receptores de IgG/metabolismo , Linfocitos T/inmunologíaRESUMEN
Tumour necrosis factor mediates chronic inflammatory pathologies including those affecting the central nervous system, but non-selective tumour necrosis factor inhibitors exacerbate multiple sclerosis. In addition, TNF receptor SF1A, which encodes one of the tumour necrosis factor receptors, has recently been identified as a multiple sclerosis susceptibility locus in genome-wide association studies in large patient cohorts. These clinical data have emphasized the need for a better understanding of the beneficial effects of tumour necrosis factor during central nervous system inflammation. In this study, we present evidence that the soluble and transmembrane forms of tumour necrosis factor exert opposing deleterious and beneficial effects, respectively, in a multiple sclerosis model. We compared the effects, in experimental autoimmune encephalomyelitis, of selectively inhibiting soluble tumour necrosis factor, and of both soluble and transmembrane tumour necrosis factor. Blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected mice against the clinical symptoms of experimental autoimmune encephalomyelitis. Therapeutic benefit was independent of changes in antigen-specific immune responses and focal inflammatory spinal cord lesions, but was associated with reduced overall central nervous system immunoreactivity, increased expression of neuroprotective molecules, and was dependent upon the activity of neuronal nuclear factor-κB, a downstream mediator of neuroprotective tumour necrosis factor/tumour necrosis factor receptor signalling, because mice lacking IκB kinase ß in glutamatergic neurons were not protected by soluble tumour necrosis factor blockade. Furthermore, blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected neurons in astrocyte-neuron co-cultures against glucose deprivation, an in vitro neurodegeneration model relevant for multiple sclerosis, and this was dependent upon contact between the two cell types. Our results show that soluble tumour necrosis factor promotes central nervous system inflammation, while transmembrane tumour necrosis factor is neuroprotective, and suggest that selective inhibition of soluble tumour necrosis factor may provide a new way forward for the treatment of multiple sclerosis and possibly other inflammatory central nervous system disorders.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , FN-kappa B/inmunología , Neuronas/metabolismo , Fármacos Neuroprotectores/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Técnicas de Cocultivo , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Etanercept , Femenino , Glicoproteínas/inmunología , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunoglobulina G/uso terapéutico , Inmunosupresores/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito , Neuronas/citología , Fragmentos de Péptidos/inmunología , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.
Asunto(s)
Sitios de Unión de Anticuerpos , Comunicación Celular/inmunología , Inmunidad Humoral , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/metabolismo , Animales , Antígenos CD19/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/patología , Comunicación Celular/genética , Modelos Animales de Enfermedad , Femenino , Amplificación de Genes/inmunología , Células HEK293 , Humanos , Inmunidad Humoral/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/trasplante , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/prevención & control , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones SCID , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de IgG/deficiencia , Receptores de IgG/fisiologíaRESUMEN
CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40(+) malignancies.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD40/inmunología , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Receptores de IgG/inmunología , Animales , Línea Celular Tumoral , Proliferación Celular , Humanos , Inmunoterapia , Leucemia/inmunología , Leucemia/terapia , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Leucemia de Células Plasmáticas/inmunología , Leucemia de Células Plasmáticas/terapia , Linfoma/inmunología , Linfoma/terapia , Ratones , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Células Tumorales CultivadasRESUMEN
CD19, a B cell-restricted receptor critical for B-cell development, is expressed in most B-cell malignancies. The Fc-engineered anti-CD19 antibody, XmAb5574, has enhanced Fcgamma receptor (FcgammaR) binding affinity, leading to improved FcgammaR-dependent effector cell functions and antitumor activity in murine xenografts compared with the non-Fc-engineered anti-CD19 IgG1 analog. Here, we use XmAb5574 and anti-CD19 IgG1 to further dissect effector cell functions in an immune system closely homologous to that of humans, the cynomolgus monkey. XmAb5574 infusion caused an immediate and dose-related B-cell depletion in the blood (to <10% of baseline levels) concomitant with a sustained reduction of natural killer (NK) cells. NK cells had fully recovered by day 15, whereas B-cell recovery was underway by day 57. B cells in secondary lymphoid tissues were depleted (to 34%-61% of vehicle), with involuted germinal centers apparent in the spleen. Anti-CD19 IgG1 had comparable serum exposure to XmAb5574 but demonstrated no B-cell depletion and no sustained NK-cell reduction. Thus, increasing FcgammaR binding affinity dramatically increased B-cell clearing. We propose that effector cell functions, possibly those involving NK cells, mediate XmAb5574 potency in cynomolgus monkeys, and that enhancing these mechanisms should advance the treatment of B-cell malignancies in humans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Ingeniería de Proteínas , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Antígenos CD19/genética , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Células Asesinas Naturales/inmunología , Depleción Linfocítica/métodos , Macaca fascicularis , Receptores de IgG/genéticaRESUMEN
CD19 is a pan B-cell surface receptor expressed from pro-B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non-Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcgamma receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcgamma receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19(+) hematologic malignancies.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Leucemia/terapia , Linfoma/terapia , Animales , Anticuerpos Monoclonales/biosíntesis , Antineoplásicos/uso terapéutico , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/química , Inmunoterapia , Leucemia/inmunología , Linfoma/inmunología , Ratones , Ratones Noqueados , Ratones SCID , Unión Proteica , Ingeniería de Proteínas/métodos , Receptores de IgG/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.
Asunto(s)
Anticuerpos/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/inmunología , Anticuerpos/genética , Apoptosis , Linfocitos B/citología , Linfocitos B/metabolismo , Calcio/metabolismo , Línea Celular , Proliferación Celular , Humanos , Inositol Polifosfato 5-Fosfatasas , Activación de Linfocitos , Monoéster Fosfórico Hidrolasas/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/genética , Transducción de Señal , Resonancia por Plasmón de SuperficieRESUMEN
A unique property of monoclonal antibodies, and a principal reason for their success as cancer therapeutics, is their ability to engage the immune system. A growing set of data supporting the relevance of Fc-mediated effector functions to anti-tumor efficacy has motivated efforts to enhance the interactions between antibodies and Fc receptors expressed on immune cells. Although current approaches have considerable promise for improved clinical performance, the immunobiology of tumors, antibodies, and Fc receptors continues to evolve. In this review we discuss what is known and what is not known about the interactions between therapeutic antibodies and the immune system, with the goal being progress toward clear target profiles for effector engineering efforts.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/inmunología , Sistema Inmunológico/fisiología , Neoplasias/terapia , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Citocinas/fisiología , Humanos , Neoplasias/inmunología , Ingeniería de Proteínas , Receptores de IgG/genética , Receptores de IgG/inmunología , Receptores de IgG/fisiologíaRESUMEN
The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.