RESUMEN
MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023's binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults.
Asunto(s)
Hormona de Crecimiento Humana/farmacocinética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fragmentos de Péptidos/química , Receptores de Somatotropina/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Western Blotting , Proliferación Celular , Femenino , Semivida , Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/química , Humanos , Técnicas In Vitro , Macaca mulatta , Masculino , Ratones , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-beta-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.
Asunto(s)
Gonadotropina Coriónica Humana de Subunidad beta/genética , Hormona de Crecimiento Humana/genética , Sistemas de Lectura Abierta/genética , Proteínas Recombinantes de Fusión/genética , Animales , Área Bajo la Curva , Western Blotting , Células CHO , Gonadotropina Coriónica Humana de Subunidad beta/química , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Cricetinae , Cricetulus , Hormona de Crecimiento Humana/metabolismo , Humanos , Hipofisectomía , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Factores de Tiempo , Aumento de Peso/efectos de los fármacosRESUMEN
Many viruses escape the cellular immune response by downregulating cell surface expression of major histocompatibility complex (MHC) class I molecules. However, infection of cells with flaviviruses can upregulate the expression of these molecules. In this study we analyzed the expression of MHC class I in K562 and THP-1 human cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and express all the dengue virus nonstructural proteins together. We show that MHC class I expression is upregulated in the dengue virus replicon-expressing cells and that the binding of natural killer (NK) inhibitory receptors to these cells is augmented. This upregulation results in reduced susceptibility of the dengue virus replicon-expressing cells to NK lysis, indicating a possible mechanism for evasion of the dengue virus from NK cell recognition. Visualizing MHC class I expression in replicon-containing K562 and THP-1 cells by confocal microscopy demonstrated aggregation of MHC class I molecules on the cell surface. Finally, replicon-expressing K562 cells manifested increased TAP (transporter associated with antigen processing) and LMP (low-molecular-mass protein) gene transcription, while replicon-expressing THP-1 cells manifested increased NF-kappaB activity and MHC class I transcription. We suggest that expression of dengue virus nonstructural proteins is sufficient to induce MHC class I upregulation through both TAP-dependent and -independent mechanisms. Additionally, aggregation of MHC class I molecules on the cell membrane also contributes to significantly higher binding of low-affinity NK inhibitory receptors, resulting in lower sensitivity to lysis by NK cells.
Asunto(s)
Citotoxicidad Inmunológica , Virus del Dengue/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Células Asesinas Naturales/inmunología , Proteínas no Estructurales Virales/inmunología , Transportadoras de Casetes de Unión a ATP/biosíntesis , Línea Celular , Membrana Celular/química , Pruebas Inmunológicas de Citotoxicidad , Humanos , Microscopía Confocal , FN-kappa B/metabolismoRESUMEN
The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.
Asunto(s)
Inmunoterapia , Células Asesinas Naturales/inmunología , Neoplasias de la Próstata/terapia , Receptores Inmunológicos/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones SCIDRESUMEN
Human melanoma cell lines were shown to express ligands for the natural cytotoxicity receptor, NKp46, expressed by natural killer (NK) cells. We aimed to examine the expression of ligands for NKp46 by various primary human melanocytic cells and melanocytic lesions. Sections from primary nevi and melanomas were tested for expression of NKp46 ligands employing chimeric NKp46-Fc for staining. The melanocytes present in the reticular dermis were negative for NKp46 ligands in common nevi; in malignant melanocytic lesions, the deeper melanocytes were focally positive. In dermoepidermal junction of all melanocytic lesions, the melanocytes showed enhanced expression of NKp46 ligands. Melanophages in all lesions were consistently positive for NKp46 ligands. These observations establish the expression of NKp46 ligands by primary-transformed melanocytes. Normal melanocytes did not express ligands to NKp46. Therefore, the results show (i) a correlation between the malignant potential of the lesion and the expression of NKp46 ligands in the reticular dermis, and (ii) enhanced expression of NKp46 ligands in the active proliferation zone (dermoepidermal junction) of nevi and melanomas. Ligands to NKp46 were expressed on the membrane and within the cells. The physiological role of NKp46 ligands in the progression of malignancy within melanocytic lesions should be explored further.
Asunto(s)
Melanoma/inmunología , Melanoma/patología , Nevo/inmunología , Nevo/patología , Receptores Inmunológicos/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Línea Celular Tumoral , Membrana Celular/inmunología , Dermis/inmunología , Dermis/patología , Progresión de la Enfermedad , Humanos , Células Asesinas Naturales/inmunología , Ligandos , Melanocitos/inmunología , Melanocitos/patología , Receptor 1 Gatillante de la Citotoxidad NaturalRESUMEN
NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.
Asunto(s)
Heparitina Sulfato/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Sitios de Unión , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Glicosilación , Células HeLa , Humanos , Receptor 3 Gatillante de la Citotoxidad Natural , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H5N1 del Virus de la Influenza A/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/metabolismo , Línea Celular , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Células Asesinas Naturales/virología , Receptor 2 Gatillante de la Citotoxidad Natural , Receptores Inmunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.
Asunto(s)
Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Células CHO , Carcinoma Ductal/patología , Línea Celular Tumoral , Cricetinae , Cricetulus , Epítopos , Células HeLa , Heparitina Sulfato/metabolismo , Humanos , Inmunoglobulinas/genética , Interferón gamma/metabolismo , Ligandos , Masculino , Melanoma/patología , Datos de Secuencia Molecular , Mutación , Receptor 2 Gatillante de la Citotoxidad Natural , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/patología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.
Asunto(s)
Anticoagulantes/metabolismo , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/química , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Heparina , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Receptor 1 Gatillante de la Citotoxidad Natural , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Alineación de SecuenciaRESUMEN
Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.
Asunto(s)
Carbohidratos/inmunología , Proteoglicanos de Heparán Sulfato/inmunología , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/inmunología , Receptores Inmunológicos , Animales , Línea Celular Tumoral , Citometría de Flujo , HumanosRESUMEN
The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.
Asunto(s)
Acetolactato Sintasa/química , Arginina/química , Acetolactato Sintasa/metabolismo , Aniones , Benzaldehídos/farmacología , Sitios de Unión , Butiratos/farmacología , Carbono/química , Catálisis , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Secuencia Conservada , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Escherichia coli/enzimología , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
The destruction of viral-infected and tumor cells is mediated in part via the lysis receptor of natural killer (NK) cells, NKp46. The nature, however, of its lysis ligands expressed on target cells is poorly defined. Recently, we have identified a novel functional interaction between the lysis receptors NKp46 and NKp44 and the hemagglutinin of influenza and hemagglutinin-neuroaminidase of Sendai viruses. This recognition depends on the sialylation of NKp46 and NKp44 receptors. In this study, we expand the significance of these observations by demonstrating a conserved pattern of NKp46 and NKp44 recognition by various hemagglutinins derived from different viral strains. We further establish that this recognition is direct and mainly mediated via alpha2,6-linked sialic acid carried by NKp46. In addition, we demonstrate that the ability of NKp46 to recognize target cells is confined to the membrane proximal domain, and largely relies on the highly conserved sugar-carrying residue, Thr 225. This residue plays a critical dual role in NKp46 interactions with both viral hemagglutinins and the unknown tumor ligands via different mechanisms. These results may explain the ability of NK cells to kill such a broad spectrum of viral-infected and tumor cells.