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1.
J Immunol Methods ; 371(1-2): 122-33, 2011 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-21756911

RESUMO

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.


Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Cadeias kappa de Imunoglobulina/isolamento & purificação , Imunotoxinas/isolamento & purificação , Fatores de Virulência/imunologia , ADP Ribose Transferases/uso terapêutico , Animais , Toxinas Bacterianas/uso terapêutico , Linhagem Celular , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Exotoxinas/uso terapêutico , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/uso terapêutico , Imunotoxinas/química , Imunotoxinas/uso terapêutico , Camundongos , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Fatores de Virulência/uso terapêutico , Exotoxina A de Pseudomonas aeruginosa
2.
J Immunol ; 185(7): 3990-4003, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20739677

RESUMO

Depletion of CD20(+) B cells has been related to reduced clinical activity in relapsing-remitting multiple sclerosis. The underlying mechanism is not understood, because serum IgG levels were unaltered by the treatment. We report the effect of late B cell depletion on cellular and humoral immune mechanisms in a preclinical multiple sclerosis model (i.e., experimental autoimmune encephalomyelitis [EAE] in the common marmoset). We used a novel human anti-human CD20 IgG1κ mAb (HuMab 7D8) that cross-reacts with marmoset CD20. EAE was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein (MOG) in CFA. After 21 d, B cells were depleted in seven monkeys by HuMab 7D8, and seven control monkeys received PBS. The Ab induced profound and long-lasting B cell depletion from PBMCs and lymphoid organs throughout the observation period of 106 d. Whereas all of the control monkeys developed clinically evident EAE, overt neurologic deficits were reduced substantially in three HuMab 7D8-treated monkeys, and four HuMab 7D8-treated monkeys remained completely asymptomatic. The effect of HuMab 7D8 was confirmed on magnetic resonance images, detecting only small lesions in HuMab 7D8-treated monkeys. The infusion of HuMab 7D8 arrested the progressive increase of anti-MOG IgG Abs. Although CD3(+) T cell numbers in lymphoid organs were increased, their proliferation and cytokine production were impaired significantly. Most notable were the substantially reduced mRNA levels of IL-7 and proinflammatory cytokines (IL-6, IL-17A, IFN-γ, and TNF-α). In conclusion, B cell depletion prevents the development of clinical and pathological signs of EAE, which is associated with impaired activation of MOG-reactive T cells in lymphoid organs.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Depleção Linfocítica/métodos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Linfócitos B/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Callithrix , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Cadeias kappa de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/uso terapêutico , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Reação em Cadeia da Polimerase
3.
Hum Antibodies ; 18(4): 127-37, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19996527

RESUMO

Pritumumab is a human IgG1 kappa antibody that has been derived from a B-cell isolated from a regional draining lymph node of a patient with cervical carcinoma. Specificity analysis of the antibody with human tissues showed the antigen, altered tumor-associated vimentin, to be highly restricted to various cancers and not normal cells and tissues. In various clinical trials in Japan 249 patients with brain cancer were treated with pritumumab. The overall response rate was between 25-30% with several survivors beyond 5-years post-treatment. The patients were on a low dose regimen of 1mg given twice a week for a course of 24 weeks for a total dose of 48 mgs per course. Pritumumab appears to be a safe and effective therapy in patients with malignant gliomas.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Encefálicas/terapia , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Feminino , Glioma/imunologia , Glioma/patologia , Glioma/terapia , Humanos , Imunoconjugados/uso terapêutico , Imunoglobulina G/uso terapêutico , Cadeias kappa de Imunoglobulina/uso terapêutico , Japão , Masculino , Camundongos , Camundongos Nus , Transplante Heterólogo
4.
J Biol Chem ; 283(2): 899-907, 2008 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-17991752

RESUMO

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.


Assuntos
Anticorpos Catalíticos/uso terapêutico , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Catalíticos/genética , Biópsia , Infecções por Helicobacter/patologia , Helicobacter pylori/enzimologia , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/uso terapêutico , Cadeias kappa de Imunoglobulina/uso terapêutico , Camundongos , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , RNA Mensageiro/genética , Gastropatias/imunologia , Gastropatias/microbiologia , Gastropatias/patologia , Urease/química , Urease/genética
6.
Infect Immun ; 70(2): 612-9, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11796590

RESUMO

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies. Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Escherichia coli O157/imunologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Imunização Passiva , Cadeias kappa de Imunoglobulina/uso terapêutico , Toxina Shiga II/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Modelos Animais de Doenças , Feminino , Vida Livre de Germes , Células HeLa , Humanos , Imunização Passiva/métodos , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Isotipos de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Camundongos , Testes de Neutralização , Suínos
7.
Proc Natl Acad Sci U S A ; 84(22): 8100-4, 1987 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3120185

RESUMO

A murine monoclonal antibody (mAb) against porcine von Willebrand factor (vWF) induced an antithrombotic state in normal pigs. Thrombosis was induced by a standard procedure of stenosis and mechanical injury of the artery. The mAb was an IgG1 kappa that inhibited vWF-induced platelet aggregation at a titer of 1:6250 and bound to immobilized vWF at a maximal dilution of 1:512,000. The antibody did not affect two other vWF functions, platelet adhesion and binding of coagulant factor VIII (factor VIII:C). The antithrombotic state was characterized by a prolonged bleeding time and lack of plasma vWF activity, but with near-normal levels of factor VIII:C and von Willebrand antigen. The circulating Ag.mAb complex demonstrated a multimeric distribution comparable to that of native plasma vWF. Three groups of pigs were studied: group A consisted of nine untreated animals, eight of which developed occlusive coronary thrombosis; group B, four treated animals with a long bleeding time, none of which developed occlusive thrombosis; and group C, two animals with preexisting thrombosis treated with mAb, in which stable blood flow was reestablished. Morphologically, the group B animals showed adherent platelets covering the injured intima but no thrombosis. This mAb is an antithrombotic agent that prevents platelet thrombosis without affecting intrinsic platelet function.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Doença das Coronárias/prevenção & controle , Trombose Coronária/prevenção & controle , Fibrinolíticos/uso terapêutico , Fator de von Willebrand/imunologia , Animais , Tempo de Sangramento , Circulação Coronária , Trombose Coronária/tratamento farmacológico , Trombose Coronária/fisiopatologia , Imunoglobulina G/uso terapêutico , Cadeias kappa de Imunoglobulina/uso terapêutico , Camundongos , Agregação Plaquetária , Suínos
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