RESUMEN
The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.
Asunto(s)
5'-Nucleotidasa/química , 5'-Nucleotidasa/inmunología , Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Neoplasias Pulmonares/inmunología , 5'-Nucleotidasa/metabolismo , Dominio Catalítico , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Conformación Proteica , Células Tumorales CultivadasRESUMEN
Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Distroglicanos/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Modelos Animales de Enfermedad , Distroglicanos/inmunología , Expresión Génica , Humanos , Inmunohistoquímica , Inyecciones Intramusculares , Laminina/genética , Laminina/inmunología , Ratones , Ratones Noqueados , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamiento farmacológico , Síndrome de Walker-Warburg/etiologíaRESUMEN
We report a modular approach to synthesize maleimido group containing hydrophilic dolastatin 10 (Dol10) derivatives as drug-linkers for the syntheses of antibody-drug conjugates (ADCs). Discrete polyethylene glycol (PEG) moieties of different chain lengths were introduced as part of the linker to impart hydrophilicity to these drug linkers. The synthesis process involved construction of PEG maleimido derivatives of the tetrapeptide intermediate (N-methylvaline-valine-dolaisoleucine-dolaproine), which were subsequently coupled with dolaphenine to generate the desired drug linkers. The synthetic method reported in this manuscript circumvents the use of highly cytotoxic Dol10 in its native form. By using trastuzumab (Herceptin®) as the antibody we have synthesized Dol10 containing ADCs. The presence of a discrete PEG chain in the drug linkers resulted in ADCs free from aggregation. The effect of PEG chain length on the biological activities of these Dol10 containing ADCs was investigated by in vitro cytotoxicity assays. ADCs containing PEG6 and PEG8 spacers exhibited the highest level of in vitro anti-proliferative activity against HER2-positive (SK-BR-3) human tumor cells. ADCs derived from Herceptin® and PEG8-Dol10, at a dose of 10 mg kg-1, effectively delayed the tumor growth and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Depsipéptidos/farmacología , Inmunoconjugados/efectos de los fármacos , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Depsipéptidos/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Ratones SCID , Conformación Molecular , Células Tumorales CultivadasRESUMEN
Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos/química , Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados/química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glicosilación , Humanos , Ratones , Ratones SCID , Estructura Molecular , Neoplasias Experimentales/patología , Polisacáridos/química , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Relación Estructura-Actividad , TrastuzumabRESUMEN
Lysosomal storage diseases (LSDs) are metabolic disorders caused by enzyme deficiencies that lead to lysosomal accumulation of undegraded substrates. Enzyme replacement therapies (ERT) have been developed as treatments for patients with Gaucher, Niemann-Pick, Fabry, and Pompe diseases. Depending on the disease, the corresponding therapeutic enzyme is designed to be internalized by diseased cells through receptor-mediated endocytosis via macrophage mannose receptors (MMR) or mannose-6-phosphate receptors (M6PR). Enzymes developed to treat Gaucher and Niemann-Pick diseases are meant to target MMR-expressing cells, and in the case of Cerezyme [recombinant human ß-glucocerebrosidase (rhßGC)] for treating Gaucher disease, glycans on the enzyme are modified to increase specificity toward this receptor. Due to heterogeneity in glycosylation on enzymes intended to target the M6PR, however, there may also be some unintended targeting to MMR-expressing cells, which could act as unwanted sinks. Examples include Fabrazyme [recombinant human α-galactosidase A (rhαGal)] for treating Fabry disease and Myozyme [recombinant human acid α-glucosidase (rhGAA)] for treating Pompe disease. It is therefore of great interest to better understand the cell type and tissue distribution of MMR in murine LSD models used to evaluate ERT efficacy and mechanism of action. In this study, we generated affinity-purified polyclonal antibody against murine MMR and used it to carry out a systematic examination of MMR protein localization in murine models of Gaucher, Niemann-Pick, Fabry, and Pompe diseases. Using immunohistochemistry, immunofluorescence, and confocal microscopy, we examined MMR distribution in liver, spleen, lung, kidney, heart, diaphragm, quadriceps, and triceps in these animal models and compared them with MMR distribution in wild-type mice.
Asunto(s)
Lectinas Tipo C/análisis , Lectinas Tipo C/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lectinas de Unión a Manosa/análisis , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/metabolismo , Animales , Anticuerpos , Formación de Anticuerpos , Línea Celular , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Humanos , Hígado/metabolismo , Hígado/patología , Enfermedades por Almacenamiento Lisosomal/patología , Macrófagos/metabolismo , Receptor de Manosa , Ratones , Ratones Noqueados , Terapia Molecular Dirigida/métodos , Músculos/metabolismo , Músculos/patología , Bazo/metabolismo , Bazo/patología , Distribución TisularRESUMEN
Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.
RESUMEN
A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody-drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well-defined ADCs, whose DARs could be accurately determined by LC-MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatinâ 10 (MMAD). The inâ vitro potencies of the MDL-derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody-drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.
Asunto(s)
Antineoplásicos Inmunológicos/química , Inmunoconjugados/química , Polietilenglicoles/química , Trastuzumab/química , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Polietilenglicoles/farmacología , Agregado de Proteínas , Solubilidad , Trastuzumab/farmacologíaRESUMEN
BACKGROUND: Acute lung injury is a common cause of morbidity and mortality following pulmonary or systemic infections. Surfactant protein-D is a member of the collectin family of proteins, which play important roles in innate host defense of the lung. In this study, the effect of exogenous recombinant human SP-D (rhSP-D) on protection of the adult mouse lung from lipopolysaccharide (LPS)-induced and lipoteichoic acid (LTA)-induced injury was assessed. METHODS: The effect of rhSP-D on LPS-induced and LTA-induced lung inflammation and injury was assessed with and without exogenous pulmonary surfactant in Sftpd+/+ and Sftpd-/- mice. A total of 204 mice (6 mice per group) were used for the present study. RESULTS: Sftpd-/- mice were more susceptible to intratracheal LPS than were Sftpd+/+ mice. rhSP-D decreased neutrophilic infiltrates induced by LPS and LTA in the lungs of both Sftpd+/+ and Sftpd-/- mice. The addition of exogenous pulmonary surfactant to rhSP-D further decreased LPS-induced and LTA-induced pulmonary inflammation in Sftpd-/- and Sftpd+/+ mice. CONCLUSIONS: Intratracheal rhSP-D inhibited inflammation induced by intratracheal LPS and LTA instillation in the lung. The antiinflammatory effects of rhSP-D were enhanced by the addition of pulmonary surfactant, providing a potential therapy for the treatment of lung inflammation.
Asunto(s)
Inflamación/inmunología , Pulmón/metabolismo , Proteína D Asociada a Surfactante Pulmonar/inmunología , Surfactantes Pulmonares/inmunología , Enfermedad Aguda , Análisis de Varianza , Animales , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Instilación de Medicamentos , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Ratones , Ácidos Teicoicos/administración & dosificación , Ácidos Teicoicos/farmacologíaRESUMEN
Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/genética , Neoplasias/enzimología , Proteínas y Péptidos Salivales/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas y Péptidos Salivales/análisis , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia ArribaRESUMEN
Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines. Examination of the CNS revealed a decrease in infiltrating lymphocytes, demyelination and axonal loss. Electrophysiological assessment showed preservation of axonal conductance in the spinal cord. These findings suggest that anti-CD52 therapy may help preserve CNS integrity.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Glicoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Axones/efectos de los fármacos , Axones/inmunología , Axones/patología , Antígeno CD52 , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/patología , Glicoproteínas/antagonistas & inhibidores , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.
Asunto(s)
Bioensayo , Proteínas de la Membrana/metabolismo , Resonancia por Plasmón de Superficie , Anticuerpos/metabolismo , Afinidad de Anticuerpos , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Bioensayo/métodos , Antígeno CD52 , Glicoproteínas/metabolismo , Humanos , Cinética , Ligandos , Proteínas de la Membrana/química , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Receptores CXCR5/química , Receptores CXCR5/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Resonancia por Plasmón de Superficie/métodosRESUMEN
Choriocarcinomas are a rare form of cancer that develops in the uterus from tissue which would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and have been studied largely to investigate conditions related to pregnancy such as preeclampsia. Choriocarcinomas are highly angiogenic and produce high levels of placental growth factor (PlGF) to promote the development of blood vessels. Upregulation of PlGF expression also occurs during the development of other human malignancies such as breast cancer and melanoma. Both tumor specimens and plasma samples have higher levels of PlGF than normal tissues. Hence, PlGF has emerged as a valid target for anti-angiogenic therapy. The cell lines BeWo, JAR and JEG-3, derived from human choriocarcinomas, were investigated in vitro and in vivo for suitability as PlGF-dependent models. BeWo, JAR and JEG-3 cells were characterized in culture and were implanted into immunodeficient mice to generate subcutaneous tumors. The PlGF and VEGF angiogenic profiles of the choriocarcinoma cells and tumors were investigated by ELISA and by immunohistochemical methods. Double immunofluorescence methods were applied to choriocarcinoma xenograft sections to characterize the cellular components of the blood vessels. sFLT01, a fusion protein that neutralizes PlGF, was assessed in cell culture experiments and xenograft studies. The novel results presented here validate the importance of human choriocarcinoma cell lines and xenografts in further exploring the role of PlGF in tumor angiogenesis, for evaluating PlGF as an anti-angiogenic target, and for developing therapies that may provide clinical benefit.
Asunto(s)
Antineoplásicos/farmacología , Coriocarcinoma/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Proteínas Gestacionales/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Neoplasias Uterinas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Coriocarcinoma/irrigación sanguínea , Coriocarcinoma/patología , Medios de Cultivo Condicionados/química , Femenino , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Factor de Crecimiento Placentario , Embarazo , Proteínas Recombinantes de Fusión/uso terapéutico , Carga Tumoral , Neoplasias Uterinas/irrigación sanguínea , Neoplasias Uterinas/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. EXPERIMENTAL DESIGN: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. RESULTS: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non-tumor-bearing mice. CONCLUSION: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Proteínas Gestacionales/sangre , Sarcoma de Ewing/tratamiento farmacológico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Factor de Crecimiento Placentario , Proteínas Gestacionales/antagonistas & inhibidores , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Sarcoma de Ewing/metabolismo , Transducción de Señal , Microambiente Tumoral , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Recombinant proteins are important tools for studying biological processes. Generating a recombinant protein requires the use of an expression system. Selection of an appropriate expression system is dependent on the characteristics and intended application of the recombinant protein and is essential to produce sufficient quantities of the protein. Over the last 30 years, there have been considerable advances in the technologies for expressing recombinant proteins. In this chapter the unique characteristics of four commonly used expression systems, Escherichia coli, Pichia pastoris, baculovirus/insect cell, and mammalian cells are described. The E. coli system is a rapid method for expressing proteins but lacks many of the posttranslational modifications found in eukaryotes. The capacity of E. coli for protein folding and forming disulfide bonds is not sufficient for many recombinant proteins although there are a number of tools developed to overcome these limitations. In contrast to E. coli, the eukaryotic P. pastoris, baculovirus/insect cell, and mammalian systems promote good protein folding and many posttranslational modifications. How the characteristics and the downstream application of a recombinant protein can influence the choice of an expression system is then reviewed.
Asunto(s)
Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Recombinantes/genética , Animales , Baculoviridae/genética , Baculoviridae/crecimiento & desarrollo , Baculoviridae/metabolismo , Conducta de Elección/fisiología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Pichia/genética , Pichia/crecimiento & desarrollo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Proyectos de InvestigaciónRESUMEN
beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-glucocerebrosidase was secreted. Missorting of beta-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-glucocerebrosidase.
Asunto(s)
Antígenos CD36/metabolismo , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Manosafosfatos/metabolismo , Transporte de Proteínas , Secuencia de Aminoácidos , Animales , Antígenos CD36/genética , Línea Celular , Retículo Endoplásmico/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Glucosilceramidasa/genética , Humanos , Proteínas de Membrana de los Lisosomas/genética , Macrófagos/citología , Macrófagos/metabolismo , Manosafosfatos/genética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
RATIONALE: The susceptibility of neonates to pulmonary and systemic infection has been associated with the immaturity of both lung structure and the immune system. Surfactant protein (SP) D is a member of the collectin family of innate immune molecules that plays an important role in innate host defense of the lung. OBJECTIVES: We tested whether treatment with recombinant human SP-D influenced the response of the lung and systemic circulation to intratracheally administered Escherichia coli lipopolysaccharides. METHODS: After intratracheal lipopolysaccharide instillation, preterm newborn lambs were treated with surfactant and ventilated for 5 h. MEASUREMENT: Survival rate, physiologic lung function, lung and systemic inflammation, and endotoxin level in plasma were evaluated. MAIN RESULTS: In control lambs, intratracheal lipopolysaccharides caused septic shock and death associated with increased endotoxin in plasma. In contrast, all lambs treated with recombinant human SP-D were physiologically stable and survived. Leakage of lipopolysaccharides from the lungs to the systemic circulation was prevented by intratracheal recombinant human SP-D. Recombinant human SP-D prevented systemic inflammation and decreased the expression of IL-1beta, IL-8, and IL-6 in the spleen and liver. Likewise, recombinant human SP-D decreased IL-1beta and IL-6 in the lung and IL-8 in the plasma. Recombinant human SP-D did not alter pulmonary mechanics following endotoxin exposure. Recombinant human SP-D was readily detected in the lung 5 h after intratracheal instillation. CONCLUSIONS: Intratracheal recombinant human SP-D prevented shock caused by endotoxin released from the lung during ventilation in the premature newborn.
Asunto(s)
Proteína D Asociada a Surfactante Pulmonar/uso terapéutico , Surfactantes Pulmonares/uso terapéutico , Choque Séptico/prevención & control , Animales , Animales Recién Nacidos , Causas de Muerte , Modelos Animales de Enfermedad , Endotoxinas/sangre , Escherichia coli , Femenino , Edad Gestacional , Humanos , Interleucina-1/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Intubación Intratraqueal , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Masculino , Neumonía/etiología , Neumonía/prevención & control , Respiración Artificial , Mecánica Respiratoria/efectos de los fármacos , Ovinos , Tasa de SupervivenciaRESUMEN
The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.