RESUMEN
Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
Asunto(s)
Homeostasis/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Homeostasis/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD20/inmunología , Complejo CD3/inmunología , Leucemia de Células B/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Humanos , Leucemia de Células B/inmunología , Macaca fascicularis , Ratones , Ratones TransgénicosRESUMEN
During drug development, measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen, and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However, accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets, epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease, and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques, we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb, binding of two reagent antibodies, as required for a classic sandwich ELISA, was not feasible, and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites, and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method, total LTα could be accurately measured in cynomolgus macaque serum, and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Inmunoensayo/métodos , Linfotoxina-alfa/sangre , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacocinética , Humanos , Ligandos , Linfotoxina-alfa/inmunología , Macaca fascicularis , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Fucosa/inmunología , Células Asesinas Naturales/inmunología , Receptores de IgG/inmunología , Actinas/metabolismo , Anticuerpos Monoclonales Humanizados/química , Afinidad de Anticuerpos/inmunología , Degranulación de la Célula/inmunología , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Transducción de Señal/inmunología , Trastuzumab/química , Trastuzumab/inmunologíaRESUMEN
Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Activación de Linfocitos , Receptor ErbB-2/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratas , Ratas Sprague-Dawley , TrastuzumabRESUMEN
Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.
Asunto(s)
Artritis Experimental/inmunología , Citocinas/inmunología , Inflamación/inmunología , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Células Cultivadas , Citocinas/metabolismo , Femenino , Citometría de Flujo , Células HEK293 , Miembro Posterior/efectos de los fármacos , Miembro Posterior/inmunología , Miembro Posterior/patología , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Osteoartritis/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Biomarcadores/sangre , Factor de Crecimiento de Hepatocito/sangre , Proteínas Proto-Oncogénicas c-met/sangre , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Endocitosis , Citometría de Flujo , Células HeLa , Humanos , Inmunoensayo/métodos , Macaca fascicularis , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Microscopía Fluorescente , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/inmunología , Interferencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We characterized the pharmacokinetics of onartuzumab (MetMAb) in animals and determined a concentration-effect relationship in tumor-bearing mice to enable estimation of clinical pharmacokinetics and target doses. EXPERIMENTAL DESIGN: A tumor growth inhibition model was used to estimate tumoristatic concentrations (TSC) in mice. Human pharmacokinetic parameters were projected from pharmacokinetics in cynomolgus monkeys by the species-invariant time method. Monte Carlo simulations predicted the percentage of patients achieving steady-state trough serum concentrations (Ctrough ss) ≥TSC for every 3-week (Q3W) dosing. RESULTS: Onartuzumab clearance (CL) in the linear dose range was 21.1 and 12.2 mL/d/kg in mice and cynomolgus monkeys with elimination half-life at 6.10 and 3.37 days, respectively. The estimated TSC in KP4 pancreatic xenograft tumor-bearing mice was 15 µg/mL. Projected CL for humans in the linear dose range was 5.74 to 9.36 mL/d/kg with scaling exponents of CL at 0.75 to 0.9. Monte Carlo simulations projected a Q3W dose of 10 to 30 mg/kg to achieve Ctrough ss of 15 µg/mL in 95% or more of patients. CONCLUSIONS: Onartuzumab pharmacokinetics differed from typical bivalent glycosylated monoclonal antibodies with approximately 2-times faster CL in the linear dose range. Despite this higher CL, xenograft efficacy data supported dose flexibility with Q1W to Q3W dose regimens in the clinical setting with a TSC of 15 µg/mL as the Ctrough ss target. The projected human efficacious dose of 10 to 30 mg/kg Q3W should achieve the target TSC of 15 µg/mL. These data show effective pharmacokinetic/pharmacodynamic modeling to project doses to be tested in the clinic.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Simulación por Computador , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Macaca fascicularis , Ratones , Ratones Desnudos , Método de Montecarlo , Neoplasias Pancreáticas/metabolismo , Valor Predictivo de las Pruebas , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.
Asunto(s)
4-Aminopiridina/análogos & derivados , 4-Aminopiridina/síntesis química , Aminopiridinas/síntesis química , Benzamidas/síntesis química , TYK2 Quinasa/antagonistas & inhibidores , 4-Aminopiridina/farmacocinética , 4-Aminopiridina/farmacología , Administración Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacología , Animales , Benzamidas/farmacocinética , Benzamidas/farmacología , Disponibilidad Biológica , Cristalografía por Rayos X , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-12/metabolismo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 3/antagonistas & inhibidores , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Unión Proteica , Ratas , Factor de Transcripción STAT4/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn's disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis. METHODS: RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles. RESULTS: In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum. CONCLUSIONS: By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.
Asunto(s)
Alelos , Predisposición Genética a la Enfermedad , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Polimorfismo Genético , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/sangre , Humanos , Enfermedades Inflamatorias del Intestino/patología , Intestinos/patología , Ratones , Modelos Moleculares , Conformación Proteica , Proteolisis , Proteínas Proto-Oncogénicas/sangre , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Linfocitos/inmunología , Linfotoxina-alfa/deficiencia , Trasplante Homólogo/efectos adversos , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Linfotoxina-alfa/inmunología , Ratones , Ratones SCID , Resonancia por Plasmón de Superficie , Trasplante Homólogo/inmunologíaRESUMEN
A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/metabolismo , Ingeniería Genética , Genómica , Linfotoxina-alfa/metabolismo , Proteómica , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Hibridomas , Linfotoxina-alfa/genética , Linfotoxina-alfa/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/ß-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like ß-chain (HGF ß), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the ß-chain stimulate Met phosphorylation by pro-HGF to levels that are â¼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF ß binding to Met, resulting in a K(D)(app) of 1.6 µm, only 8-fold weaker than the Met/HGF ß-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like ß-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Oligopéptidos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Sustitución de Aminoácidos , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cricetinae , Cricetulus , Factor de Crecimiento de Hepatocito/genética , Humanos , Mutación Missense , Oligopéptidos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Precursores de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Receptores de Factores de Crecimiento/genética , Serina Proteasas/genética , Serina Proteasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
International surgical missions, particularly those that address patients with clefts of the lip and palate, have become increasingly common. Numerous groups have been organized to provide these services. A plastic surgeon participating in these endeavors should have full knowledge of the details involved not just for himself but to maximize safety and optimize outcomes for these patients. An understanding of the issues surrounding trip preparation, the in-country logistics, proper preoperative patient selection, and intraoperative and postoperative issues are all essential to a successful experience. In this article, the authors review and discuss lessons learned from a combined total of more than 100 international trips. Relevant literature is reviewed, and additional pearls from this body of knowledge are presented.
Asunto(s)
Labio Leporino/cirugía , Fisura del Paladar/cirugía , Misiones Médicas/organización & administración , Cirugía Plástica/organización & administración , HumanosRESUMEN
Tumor necrosis factor-superfamily (TNF-SF) members, lymphotoxin (LT)-alpha and LTbeta, are proinflammatory cytokines associated with pathology in rheumatoid arthritis. LTalpha3 homotrimers are secreted, whereas LTalpha(1)beta(2) heterotrimers are expressed on the surface of activated lymphocytes. As many TNF-SF members are actively cleaved from cell membranes, we determined whether LTalphabeta heterotrimers are also cleaved, and are biologically active in rheumatoid arthritis (RA) patients. LTalphabeta heterotrimers were detected in culture supernatants from activated human T-helper (Th) 0, Th1, and Th17 cells, together with LTalpha3 and TNFalpha. The heterotimers were actively cleaved from the cell surface by ADAM17 metalloproteinase (MMP) and MMP-8, and cleavage was inhibited by TAPI-1, a TNF-alpha converting enzyme (TACE) inhibitor. Soluble LTalphabeta was detected in serum from both normal donors and RA patients, and was elevated in synovial fluid from RA patients compared to osteoarthritis (OA) patients. Levels of LTalphabeta in RA patient synovial fluid correlated with increased TNFalpha, IL-8, IL-12, IL-1beta, IFN-gamma, and IL-6 cytokines. Moreover, recombinant LTalpha1beta2-induced CXCL1, CXCL2, IL-6, IL-8, VCAM-1, and ICAM-1 from primary synovial fibroblasts isolated from RA patients. Therefore, soluble LTalphabeta in synovial fluid is associated with a proinflammatory cytokine milieu that contributes to synovitis in RA.
Asunto(s)
Artritis Reumatoide/complicaciones , Artritis Reumatoide/enzimología , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Metaloproteasas/metabolismo , Sinovitis/complicaciones , Sinovitis/enzimología , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/sangre , Moléculas de Adhesión Celular/metabolismo , Quimiocinas/metabolismo , Demografía , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Activación de Linfocitos/inmunología , Heterotrímero de Linfotoxina alfa1 y beta2/sangre , Masculino , Persona de Mediana Edad , Solubilidad , Líquido Sinovial/metabolismo , Sinovitis/patología , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/terapia , Interleucina-17/fisiología , Depleción Linfocítica , Linfotoxina-alfa/antagonistas & inhibidores , Células TH1/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/terapia , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/inmunología , Inflamación/etiología , Ratones , Ratones Endogámicos DBARESUMEN
The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.
Asunto(s)
Adenocarcinoma/patología , Anticuerpos Monoclonales/inmunología , Neoplasias Pancreáticas/patología , Proteínas Tirosina Quinasas Receptoras/inmunología , Adenocarcinoma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Desnudos , Neovascularización Patológica/inmunología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Fosforilación , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Flow cytometry and cell-based enzyme-linked immunosorbent assays have been used to detect surface proteins on cells. A recently available electrochemiluminescent assay methodology using carbon surface electrodes built into the bottom of microwell plates can be used as an alternative to these methods and has some advantages. The carbon surface plates bind suspension cells tightly enough to allow plates to be washed using a plate washer. This eliminates the centrifugation steps typically used for washing suspension cells. For adherent cells, human umbilical vein endothelial cells can be grown, activated, and assayed in the same carbon surface plate. This eliminates the need to detach cells from tissue culture plates for analysis, making the electrochemiluminescent assay much easier to perform than a corresponding flow cytometry assay. This electrochemiluminescence technology provides a high throughput method to detect surface proteins on live cells.