RESUMEN
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
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
Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.
Asunto(s)
Anticuerpos Monoclonales/genética , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células Cultivadas , Regiones Determinantes de Complementariedad/genética , Humanos , Técnicas In Vitro , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fc-engineered antibodies in an hFcRn/Rag1(-/-) mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Animales , Semivida , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
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.