RESUMEN
Introduction: The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. Methods: By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. Results: We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Discussion: Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.
RESUMEN
Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope-Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope-Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to "snap" functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.
Asunto(s)
Anticuerpos Monoclonales/química , Cetuximab/química , Portadores de Fármacos/química , Fragmentos Fab de Inmunoglobulinas/química , Trastuzumab/química , Animales , Anticuerpos Monoclonales/inmunología , Azidas/química , Sitios de Unión , Química Clic/métodos , Femenino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Unión Proteica , Receptor ErbB-2/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
We have previously identified a cyclic peptide called meditope which binds to the central cavity of the Fab portion of cetuximab and shown that this peptide binding site can be grafted, or 'meditope-enabled', onto trastuzumab. This peptide has been shown to act as a hitch for the non-covalent attachment of imaging agents to meditope-enabled antibodies. Herein, we explore the process of grafting this peptide binding site onto M5A, an anti-CEA antibody in clinical trials for cancer diagnostics. In order to explore the contributions of the amino acids, we sequentially introduced pairs of amino acid substitutions into the Fab and then we reverse-substituted key residues in the presence of the other substitutions. We demonstrate that Pro40Thr, Gly41Asn, Phe83Ile and Thr85Asp in the light chain are sufficient to recreate the meditope binding site in M5A with single-digit micromolar affinity. We show that Pro40 abrogates peptide binding in the presence of the other 12 residue substitutions, and that the presence of all 13 substitutions does not interfere with antibody:antigen recognition. Collectively, these studies provide detailed insight for defining and fine-tuning the binding affinity of the meditope binding site within an antibody.
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Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antígeno Carcinoembrionario/inmunología , Péptidos/genética , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Sitios de Unión/genética , Antígeno Carcinoembrionario/química , Antígeno Carcinoembrionario/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Péptidos/química , Unión Proteica/genéticaRESUMEN
Functionalization of monoclonal antibodies (mAbs) requires chemical derivatization and/or genetic manipulation. Inherent in these methods are challenges with protein heterogeneity, stability and solubility. Such perturbations could potentially be avoided by using a high affinity, non-covalent intermediate to bridge the desired functionality to a stable mAb. Recently, we engineered a binding site for a peptide named "meditope" within the Fab of trastuzumab. Proximity of the meditope site to that of protein L suggested an opportunity to enhance the meditope's moderate affinity. Joined by a peptide linker, the meditope-protein L construct has a KD ~ 180 pM - a 7000-fold increase in affinity. The construct is highly specific to the engineered trastuzumab, as demonstrated by flow cytometry. Moreover, the fusion of a bulky GFP to this construct did not affect the association with cell surface antigens. Collectively, these data indicate this specific, high affinity construct can be developed to rapidly add new functionality to mAbs.
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Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/fisiología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Trastuzumab/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Línea Celular , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Microscopía Fluorescente , Péptidos/inmunología , Resonancia por Plasmón de Superficie , Trastuzumab/genética , Trastuzumab/inmunologíaRESUMEN
Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent "linker" for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.
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Anticuerpos Monoclonales/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular Tumoral , Cetuximab , Cristalografía por Rayos X , Receptores ErbB/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptidos/química , Péptidos/genética , Unión Proteica/inmunología , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Receptor ErbB-2/inmunología , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , TrastuzumabRESUMEN
Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.
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Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/fisiología , Neoplasias/genética , Animales , Endonucleasas de ADN Solapado/metabolismo , Humanos , RatonesRESUMEN
BACKGROUND: Veterinary drugs such as clenbuterol (CL) and sulfamethazine (SM2) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique. RESULTS: The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues. CONCLUSIONS: The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.
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Clenbuterol/análisis , Residuos de Medicamentos/análisis , Análisis por Matrices de Proteínas/métodos , Sulfametazina/análisis , Animales , Pollos , Ensayo de Inmunoadsorción Enzimática , Concentración 50 Inhibidora , Músculo Esquelético/química , Ovalbúmina/química , Conejos , Sensibilidad y Especificidad , Drogas Veterinarias/análisisRESUMEN
BACKGROUND: Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells. METHODS: siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice. The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-beta-galactosidase staining. RESULTS: The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 microM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 microM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments. CONCLUSION: The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.
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Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Doxorrubicina/administración & dosificación , Terapia Genética , ARN Interferente Pequeño/uso terapéutico , Telomerasa/genética , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Terapia Combinada , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Interferente Pequeño/genética , Telomerasa/metabolismoRESUMEN
Inhibition of p38 MAPK suppresses the expression of proinflammatory cytokines such as TNF-alpha and IL-1 beta in macrophages and fibroblast-like synoviocytes (FLS). However, there have been no genomewide studies on the gene targets of p38 MAPK signaling in synoviocytes. Microarray technology was applied to generate a comprehensive analysis of all genes regulated by the p38 MAPK signaling pathway in FLS. Gene expression levels were measured with Agilent oligonucleotide microarrays. Four independent sets of mRNA modulated by TNF-alpha and vehicle were used to measure the change of gene expression due to TNF-alpha, and three experiments were done to ascertain the effect of SB-203580, a p38 MAPK inhibitor, on TNF-alpha-induced genes. Microarray data were validated by RT-quantitative polymerase chain reaction. One hundred forty-one significantly expressed genes were more than twofold upregulated by TNF-alpha. Thirty percent of these genes were downregulated by the p38 inhibitor SB-203580, whereas 67% of these genes were not significantly changed. The SB-203580-inhibited genes include proinflammatory cytokines such as interleukins and chemokines, proteases including matrix metallopeptidases, metabolism-related genes such as cyclooxygenases and phosphodiesterase, genes involved in signal transduction, and genes encoding for transcription factors, receptors, and transporters. Approximately one-third of the TNF-alpha-induced genes in FLS are regulated by the p38 MAPK signal pathway, showing that p38 MAPK is a possible target for suppressing proinflammatory gene expressions in rheumatoid arthritis.
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Regulación de la Expresión Génica/efectos de los fármacos , Membrana Sinovial/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Quimiocina CXCL2/biosíntesis , Quimiocina CXCL2/genética , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Piridinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The gastric entero-chromaffin-like (ECL) cell plays a key regulatory role in peripheral regulation of acid secretion due to the release of histamine that stimulates acid secretion by the parietal cell. Studies in intact animals, gastric glands, and isolated cells after short-term culture have shown expression of stimulatory CCK2 and PAC1 and inhibitory SST2 and Gal1 receptors as well as histidine decarboxylase. However, the pattern of its gene expression as a neuroendocrine cell has not been explored. Comparison of gene expression by 95% pure ECL cells obtained by density gradient, elutriation, and fluorescence-assisted cell sorting with isolates of the intact fundic gastric epithelium (i.e., "subtractive hybridization") identified a variety of additional expressed gene families characteristic of this neuroendocrine cell. These include genes 1) involved in neuropeptide synthesis and secretory vesicle exocytosis, 2) involved in control of inflammation, 3) implicated in healing of the epithelium, 4) encoding inhibitory Gi protein-coupled receptors, 5) playing a role in neuroendocrine regulation of food intake, and 6) encoding proteins likely involved in maintenance of circadian rhythm, in addition to the ECL cell-specific genes histidine decarboxylase and monoamine transporter. Particularly, the inhibitory apelin receptor gene, APJ, was highly expressed in the ECL cell preparation. Because parietal cells express apelin, immunohistochemical and functional studies showed that there is an inhibitory feed back loop between the parietal and ECL cell during gastrin stimulation, providing evidence for a novel pathway of downregulation of acid secretion due to interaction between these two cell types.
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Células Enterocromafines/metabolismo , Ácido Gástrico/metabolismo , Regulación de la Expresión Génica , Células Parietales Gástricas/metabolismo , Transcripción Genética , Animales , Apelina , Receptores de Apelina , Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Células Enterocromafines/efectos de los fármacos , Retroalimentación Fisiológica , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Gastrinas/farmacología , Perfilación de la Expresión Génica , Inmunohistoquímica , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina , Células Parietales Gástricas/efectos de los fármacos , ARN Mensajero/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Aci-reductone dioxygenases are key enzymes in the methionine salvage pathway. The mechanisms by which the expression of this important class of enzymes is regulated are poorly understood. Here we show that the expression of the mRNA encoding the yeast aci-reductone dioxygenase ADI1 is controlled post-transcriptionally by RNase III cleavage. Cleavage occurs in a large bipartite stem loop structure present in the open reading frame region of the ADI1 mRNA. The ADI1 mRNA is up-regulated in the absence of the yeast orthologue of RNase III Rnt1p or of the 5' --> 3' exonucleases Xrn1p and Rat1p. 3'-Extended forms of this mRNA, including a polycistronic mRNA ADI1-YMR010W mRNA, also accumulate in cells lacking Rnt1p, Xrn1p, and Rat1p or the nuclear exosome component Rrp6p, suggesting that these 3'-extended forms are subject to nuclear surveillance. We show that the ADI1 mRNA is up-regulated under heat shock conditions in a Rnt1p-independent manner. We propose that Rnt1p cleavage targets degradation of the ADI1 mRNA to prevent its expression prior to heat shock conditions and that RNA surveillance by multiple ribonucleases helps prevent accumulation of aberrant 3'-extended forms of this mRNA that arise from intrinsically inefficient 3'-processing signals.