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1.
Endocrinology ; 165(5)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38500360

RESUMO

Acromegaly and gigantism are disorders caused by hypersecretion of growth hormone (GH), usually from pituitary adenomas. Although somatostatin analogues (SSA), dopamine agonists, and GH receptor antagonists are important therapeutic agents, all of these have issues with their effectiveness, safety, and/or convenience of use. To overcome these, we developed a GH-specific potent neutralizing a mouse monoclonal antibody (mAb) named 13H02. 13H02 selectively bound both to human and monkey GH with high affinity, and strongly inhibited the biological activity of GH in the Nb2 rat lymphoma cell proliferation assay. In hypophysectomized/GH-supplemented rats, a single subcutaneous administration of 13H02 significantly and dose-dependently lowered the serum insulin-like growth factor-1 levels. To pursue the therapeutic potential of this antibody for acromegaly and gigantism, we humanized 13H02 to reduce its immunogenicity and applied a single amino acid mutation in the Fc region to extend its serum half-life. The resulting antibody, Hu-13H02m, also showed GH-specific neutralizing activity, similar to the parental 13H02, and showed improved binding affinity to human FcRn.


Assuntos
Acromegalia , Gigantismo , Hormônio do Crescimento Humano , Camundongos , Humanos , Feminino , Animais , Ratos , Hormônio do Crescimento Humano/farmacologia , Hormônio do Crescimento Humano/metabolismo , Acromegalia/tratamento farmacológico , Gigantismo/complicações , Gigantismo/tratamento farmacológico , Peptídeos Semelhantes à Insulina , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico
2.
PLoS One ; 13(5): e0196422, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29723247

RESUMO

Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC) as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos)). Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.


Assuntos
Anticorpos Monoclonais/imunologia , Nectinas/antagonistas & inibidores , Nectinas/imunologia , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/genética , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Macaca fascicularis , Masculino , Camundongos , Camundongos SCID , Nectinas/genética , Engenharia de Proteínas , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trombocitopenia/etiologia , Trombocitopenia/prevenção & controle , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Mol Cancer ; 12: 60, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23758976

RESUMO

BACKGROUND: Nectin-2 is a Ca(2+)-independent cell-cell adhesion molecule that is one of the plasma membrane components of adherens junctions. However, little has been reported about the involvement of Nectin-2 in cancer. METHODS: To determine the expression of Nectin-2 in cancer tissues and cancer cell lines, we performed gene expression profile analysis, immunohistochemistry studies, and flow cytometry analysis. We also investigated the potential of this molecule as a target for antibody therapeutics to treat cancers by generating and characterizing an anti-Nectin-2 rabbit polyclonal antibody (poAb) and 256 fully human anti-Nectin-2 monoclonal antibodies (mAbs). In addition, we tested anti-Nectin-2 mAbs in several in vivo tumor growth inhibition models to investigate the primary mechanisms of action of the mAbs. RESULTS: In the present study, we found that Nectin-2 was over-expressed in clinical breast and ovarian cancer tissues by using gene expression profile analysis and immunohistochemistry studies. Nectin-2 was over-expressed in various cancer cell lines as well. Furthermore, the polyclonal antibody specific to Nectin-2 suppressed the in vitro proliferation of OV-90 ovarian cancer cells, which express endogenous Nectin-2 on the cell surface. The anti-Nectin-2 mAbs we generated were classified into 7 epitope bins. The anti-Nectin-2 mAbs demonstrated antibody-dependent cellular cytotoxicity (ADCC) and epitope bin-dependent features such as the inhibition of Nectin-2-Nectin-2 interaction, Nectin-2-Nectin-3 interaction, and in vitro cancer cell proliferation. A representative anti-Nectin-2 mAb in epitope bin VII, Y-443, showed anti-tumor effects against OV-90 cells and MDA-MB-231 breast cancer cells in mouse therapeutic models, and its main mechanism of action appeared to be ADCC. CONCLUSIONS: We observed the over-expression of Nectin-2 in breast and ovarian cancers and anti-tumor activity of anti-Nectin-2 mAbs via strong ADCC. These findings suggest that Nectin-2 is a potential target for antibody therapy against breast and ovarian cancers.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/imunologia , Moléculas de Adesão Celular/imunologia , Neoplasias Ovarianas/imunologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Expressão Gênica , Humanos , Camundongos , Nectinas , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
MAbs ; 4(6): 732-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23007682

RESUMO

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Fator de Crescimento Epidérmico/imunologia , Heparina/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Vias de Administração de Medicamentos , Humanos , Hibridomas , Imunização , Camundongos , Camundongos Endogâmicos , Neoplasias/imunologia
5.
Protein Expr Purif ; 80(2): 239-45, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21745574

RESUMO

LIGHT is a membrane-bound protein that belongs to the tumor necrosis factor (TNF) superfamily ligands. In this study, we established an effective strategy for producing a bioactive soluble form of LIGHT (sLIGHT), an extracellular region (Ile84-Val²4°) of human LIGHT. Because sLIGHT was expressed as inclusion bodies in Escherichia coli, we investigated reagents that enhance the renaturation of sLIGHT from the inclusion bodies. Interestingly, L-cysteine in the denaturation buffer containing 3.5 M guanidine hydrochloride significantly improved the renaturation efficiency of sLIGHT. The effect of L-cysteine was synergistically enhanced by L-arginine in the refolding buffer. The optimal concentrations of L-cysteine and L-arginine in the denaturation and refolding buffers were 8 mM and 0.8 M, respectively. With these buffers, approximately 90 mg of sLIGHT was purified from 200 g of frozen E. coli cells. sLIGHT thus obtained significantly induced apoptosis in the WiDr human colon adenocarcinoma cell line at nanomolar concentrations, the same amount of sLIGHT that was produced by Sf9 insect cells. These results suggest that L-cysteine in the denaturation buffer enhances the renaturation of recombinant proteins from inclusion bodies in E. coli.


Assuntos
Cisteína/farmacologia , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Renaturação Proteica , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/isolamento & purificação , Alanina/farmacologia , Apoptose , Bioensaio , Soluções Tampão , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Guanidina/farmacologia , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/farmacologia
6.
Appl Microbiol Biotechnol ; 90(5): 1691-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21400099

RESUMO

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.


Assuntos
Citotoxicidade Imunológica , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Bioensaio , Linhagem Celular Tumoral , Humanos , Camundongos , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/toxicidade , Solubilidade , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/toxicidade
7.
Micron ; 35(1-2): 105-6, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15036307

RESUMO

Keyhole limpet hemocyanin (KLH) is known to share carbohydrate epitopes with Schistosoma mansoni. In order to define the structural basis for the observed serological cross-reactivity, KLH glycans were released either by enzyme treatment or by hydrazinolysis and probed with a rabbit hyperimmune serum directed against S. mansoni egg antigen. Both major, non-reacting oligosaccharide species as well as the minor compounds recognized were isolated by two-dimensional high performance liquid chromatography and in part by lectin affinity chromatography, and characterized by mass spectrometry. The results revealed that KLH carries predominantly high mannose-type glycans as well as short sugar chains. As a characteristic feature, a number of the latter glycans contained a Gal(beta1-6)Man-unit, which has not yet been found in glycoprotein-N-glycans. Oligosaccharides cross-reacting with schistosomal glycans comprised a terminal Fuc(alpha1-3)GalNAc-motif, which appears to represent the main carbohydrate epitope mediating cross-reactivity of KLH with glycoconjugates from S. mansoni.


Assuntos
Glicoconjugados/imunologia , Hemocianinas/química , Polissacarídeos/química , Schistosoma mansoni/química , Animais , Epitopos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
J Pharm Pharmacol ; 55(7): 951-61, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12906752

RESUMO

A new animal model to evaluate the long-term growth rate produced by a sustained-release formulation of recombinant human growth hormone (rhGH) over one month was developed and the usefulness of our microcapsule formulations was demonstrated in this model. Long-term pharmacological effects by subcutaneous injection of microcapsules for sustained release of rhGH were evaluated in hypophysectomized (Hpx) rats treated with immunosuppressive agent along with hormone supplement. Copoly(DL-lactic/glycolic)acid (PLGA) microcapsules for sustained release of rhGH, a two-week sustained-release formulation (rhGH-SR-2W) and a one-month sustained-release formulation (rhGH-SR-1M), were prepared by a solid-in-oil-in-water emulsion solvent evaporation technique. Body-weight gain, body-length gain and serum levels of rat insulin-like growth factor-I (rIGF-I) induced by subcutaneous injection of rhGH-SR were compared with those by daily injections of rhGH solution in Hpx rats for 35 days. Serum IGF-I levels in Hpx rats after the injection of rhGH-SR2W microcapsules were higher than those after daily injections of rhGH solution. Body-length gain, a new parameter, after single injection of rhGH-SR-1M microcapsules demonstrated the higher growth rate than that after daily injections of rhGH solution for 35 days. Thus, single injection of rhGH-SR microcapsules demonstrated long-term pharmacological effects greater than those by daily injections of rhGH solution in a newly developed model, immunosuppressed Hpx rats.


Assuntos
Crescimento/efeitos dos fármacos , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/química , Ácido Láctico/química , Modelos Animais , Ácido Poliglicólico/química , Polímeros/química , Animais , Peso Corporal/efeitos dos fármacos , Cápsulas , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Preparações de Ação Retardada , Feminino , Hormônio do Crescimento Humano/sangue , Hipofisectomia , Terapia de Imunossupressão , Injeções Subcutâneas , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-Dawley , Solubilidade
9.
J Control Release ; 88(2): 229-42, 2003 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-12628330

RESUMO

Biodegradable microcapsules for sustained release of recombinant human growth hormone (rhGH) were prepared by a solid-in-oil-in-water (S/O/W) emulsion solvent evaporation technique using lyophilized protein microparticles. The minimum mean particle size of rhGH in S/O dispersions was 2.8-3.0 microm when ammonium acetate was added at molar ratios of 10-20 times against rhGH. High entrapment of rhGH in microcapsules was achieved by incorporating rhGH powder with a smaller particle size obtained by lyophilizing with ammonium acetate. As the particle size of rhGH decreased, the in vivo initial release decreased, while subsequent serum levels of rhGH in sustained release phase were higher. Addition of zinc oxide to microcapsules resulted in higher serum levels than those prepared without zinc oxide, suggesting a stabilizing effect of zinc oxide after subcutaneous injection into rats. The release profile of rhGH from microcapsules was controllable by selecting the proper copoly(DL-lactic/glycolic)acid (PLGA) with L/G ratio and molecular weight. Utilization of rhGH powder with a smaller particle size obtained by lyophilizing with ammonium acetate is essential for preparation of microcapsules with high entrapment and well-controlled sustained release profile with small initial release.


Assuntos
Hormônio do Crescimento/administração & dosagem , Acetatos/química , Animais , Cápsulas , Preparações de Ação Retardada , Composição de Medicamentos/métodos , Emulsões , Liofilização , Hormônio do Crescimento/farmacocinética , Humanos , Injeções Subcutâneas , Masculino , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Pós , Ratos , Ratos Sprague-Dawley , Solventes , Zinco/administração & dosagem , Zinco/farmacocinética
10.
Int J Pharm ; 251(1-2): 133-41, 2003 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-12527183

RESUMO

A procedure to prepare a complex of copoly (dl-lactic/glycolic acid) and zinc oxide (PLGA-zinc oxide complex) was developed. Out of sparingly water-soluble zinc compounds, zinc oxide was most remarkably soluble in a PLGA/dichloromethane solution and the dissolution rates became faster as the water contents in the PLGA/dichloromethane solutions increased. Since the solubility of zinc oxide was saturated at approximately 0.5-fold molar ratio to PLGA and water was generated with dissolution of zinc oxide in the PLGA/dichloromethane solutions, it is suggested that zinc oxide interacts with the terminal carboxyl group of PLGA. In addition, the glass-transition temperature of a solid material obtained by vacuum-drying the PLGA/dichloromethane solution dissolving zinc oxide became higher as the zinc content increased, suggesting that the formation of a PLGA-zinc oxide complex. Microcapsules were prepared with the PLGA-zinc oxide complex using recombinant human growth hormone (rhGH) in order to evaluate an effect of the complex on protein release and stability of protein in the microcapsules. Released rhGH amount from the microcapsules prepared with the PLGA-zinc oxide complex after subcutaneous administration in rats was significantly larger than that from microcapsules prepared with PLGA alone, indicating that rhGH molecules in the microcapsules was stabilized by the PLGA-zinc oxide complex.


Assuntos
Hormônio do Crescimento Humano/síntese química , Ácido Láctico/síntese química , Ácido Poliglicólico/síntese química , Polímeros/síntese química , Proteínas Recombinantes/síntese química , Óxido de Zinco/síntese química , Animais , Cápsulas , Hormônio do Crescimento Humano/farmacocinética , Ácido Láctico/farmacocinética , Masculino , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Solubilidade , Óxido de Zinco/farmacocinética
11.
Eur J Biochem ; 269(22): 5459-73, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12423344

RESUMO

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(beta1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.


Assuntos
Hemocianinas/química , Hemocianinas/metabolismo , Polissacarídeos/química , Motivos de Aminoácidos , Animais , Carboidratos/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Fucose/química , Galactose/química , Glicopeptídeos/química , Manose/química , Metilação , Modelos Químicos , Moluscos , Oligossacarídeos/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/farmacologia
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