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1.
Cell ; 183(2): 429-441.e16, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32941803

RESUMO

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Biblioteca de Peptídeos , Pneumonia Viral/tratamento farmacológico , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , COVID-19 , Cricetinae , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/ultraestrutura , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Pandemias , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Tratamento Farmacológico da COVID-19
2.
Eur J Pharm Sci ; 122: 42-50, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29933078

RESUMO

The variable domain of the heavy chain antibodies (VHHs) is the smallest (15 kDa) intact single domain antigen-binding fragment. VHHs often exhibit sub-nanomolar affinity for their designated targets and therefore are receiving increasing attention in molecular targeting and nanomedicine engineering. We cloned and expressed four non-overlapping anti-HER2 VHHs in a prokaryotic expression system that yielded disulfide-bonded VHHs. Purified VHHs, before and after thiolation, were characterized by Western blot and their functionality against the ecto-domain of HER2 receptor was confirmed by ELISA and flow cytometry. Thiolated VHHs were conjugated to the reactive maleimide-PEG2000-distearoylphosphatidylethanolamine incorporated into the bilayer of small unilamellar vesicles. We show high target-binding avidity and efficient cytotoxicity of optimized tetra-specific multivalent methatoraxate-loaded VHH-PEG-liposomes (55-60 VHH/vesicle) in HER2 over-expressing breast carcinoma cell lines compared with the best performing monoclonal VHH conjugated vesicles of identical VHH surface density. The VHH expression and production methodology as well as the synergistic effect of the four non-overlapping VHHs in HER2 binding provides an efficient approach for design and engineering of anti-cancer nanomedicines and their future applications within the context of personalized and precision therapies and diagnostics are discussed.


Assuntos
Antineoplásicos/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Metotrexato/administração & dosagem , Receptor ErbB-2/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lipossomos , Polietilenoglicóis/administração & dosagem
3.
PLoS One ; 11(9): e0162351, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27603013

RESUMO

Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 µg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 µg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host's humoral immune response against RVA. When 2KD1 (200 µg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.


Assuntos
Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/uso terapêutico , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Animais , Animais Recém-Nascidos , Camelídeos Americanos , Diarreia/tratamento farmacológico , Diarreia/virologia , Fezes/virologia , Concentração de Íons de Hidrogênio , Imunidade Humoral/imunologia , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Intestinos/patologia , Intestinos/virologia , Camundongos Endogâmicos BALB C , Mutação/genética , Filogenia , Proteólise , Infecções por Rotavirus/imunologia , Vírion/metabolismo , Eliminação de Partículas Virais
4.
FEBS J ; 282(18): 3618-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26147692

RESUMO

UNLABELLED: Accumulation and aggregation of the amyloid-ß (Aß) peptide is associated with Alzheimer's disease (AD). Aß is generated from the amyloid precursor protein by the successive action of two membrane-associated processing enzymes: ß-secretase or ß-site of amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase. Inhibition of one or both of these enzymes prevents Aß generation and the accompanying Aß accumulation. Antigen binding fragments from camelid heavy chain only antibodies (VHHs) were found to exert excellent enzyme inhibition activity. In the present study, we generated VHHs against BACE1 by active immunization of Lama glama with the recombinant BACE1 protein. Two classes of VHHs were selected from a VHH-phage display library by competitive elution with a peptide encoding the Swedish mutation variant of the BACE1 processing site. One VHH was found to inhibit the enzyme activity of BACE1 in vitro and in cell culture, whereas two other VHHs were found to stimulate BACE1 activity under the same conditions in vitro. Furthermore, an in vivo study with a transgenic AD mouse model, using intracisternal injection of the inhibitory VHH, led to acute reduction of the Aß load in the blood and brain. This inhibitory VHH may be considered as a candidate molecule for a therapy directed towards reduction of Aß load and prevention of AD progression. Both the inhibitory and stimulatory VHH may be useful for improving our understanding of the structure-function relationship of BACE1, as well as its role in AD progression. DATABASE: The GenBank sequence accession numbers are KR363186 for VHH B1a; KR363187 for VHH B3a; and KR363188 for VHH B5a.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/imunologia , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/imunologia , Camelídeos Americanos/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Fragmentos de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinação
5.
J Control Release ; 203: 40-50, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25668771

RESUMO

Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain.


Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/antagonistas & inibidores , Encéfalo/metabolismo , Glutationa/metabolismo , Lipossomos/metabolismo , Anticorpos de Cadeia Única/administração & dosagem , Doença de Alzheimer/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Camelídeos Americanos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Humanos , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Camundongos , Camundongos Transgênicos , Polietilenoglicóis/metabolismo , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Distribuição Tecidual
6.
Lab Invest ; 94(8): 893-905, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24933423

RESUMO

The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.


Assuntos
Adenoviridae/fisiologia , Camelídeos Americanos , Antígeno Carcinoembrionário/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Região Variável de Imunoglobulina/administração & dosagem , Tropismo Viral , Animais , Especificidade de Anticorpos , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Antígeno Carcinoembrionário/química , Linhagem Celular , Linhagem Celular Tumoral , Estudos de Viabilidade , Vetores Genéticos/administração & dosagem , Humanos , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Masculino , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução Genética , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/fisiologia
7.
J Control Release ; 159(2): 281-9, 2012 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-22227023

RESUMO

The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes.


Assuntos
Antineoplásicos/administração & dosagem , Catecóis/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Receptor IGF Tipo 1/antagonistas & inibidores , Tirfostinas/administração & dosagem , Animais , Antineoplásicos/farmacologia , Ligação Competitiva , Western Blotting , Catecóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Citometria de Fluxo , Humanos , Cadeias Pesadas de Imunoglobulinas/farmacologia , Lipossomos , Camundongos , Microscopia Confocal , Células NIH 3T3 , Nanopartículas , Tamanho da Partícula , Inibidores de Proteínas Quinases/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Propriedades de Superfície , Tirfostinas/farmacologia
8.
Biotechnol Bioeng ; 108(2): 404-12, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20830675

RESUMO

Although human IgG heavy chain genes encode a C-terminal lysine, this residue is mostly absent from the endogenous antibodies isolated from serum. Some low but variable level of C-terminal lysine is present on therapeutic antibodies expressed in mammalian cell culture systems. Here, we monitored the C-terminal lysine processing of a recombinant human IgG2 antibody after intravenous injection into human subjects. Peptide mapping of the therapeutic antibody isolated from serum samples by affinity purification was used to quantify the C-terminal lysine levels over time in vivo. The C-terminal lysine residue was found to be rapidly lost in vivo with a half life of about an hour (62 min). In vivo C-terminal lysine processing could be reproduced in vitro, but at a faster rate, by incubating in human serum. Pretreated serum, under conditions used to inactivate carboxypeptidase U, generated in vitro C-terminal lysine processing rates that more closely matched those in vivo. Endogenous IgG, isolated from human blood, contained very low levels of C-terminal lysine (∼0.02%), consistent with the expected circulating half life of antibodies and the calculated C-terminal lysine processing rate. Thus, the low residual IgG2 C-terminal lysine is rapidly processed in vivo and such processing likely occurs on endogenous antibodies in circulation.


Assuntos
Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Lisina/metabolismo , Meia-Vida , Experimentação Humana , Humanos , Imunoglobulina G/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética
9.
Brain Res ; 1369: 203-7, 2011 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-21047502

RESUMO

Erythropoietin (EPO) is a potential new treatment for acute stroke. However, EPO does not cross the blood-brain barrier (BBB). EPO has been re-engineered as an IgG-EPO fusion protein, where EPO is fused to the heavy chain of a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), which is designated the cTfRMAb-EPO fusion protein. The re-engineered EPO is able to penetrate the BBB following intravenous (IV) administration owing to transport on the BBB TfR. In the present study, the neuroprotective properties of EPO alone and the cTfRMAb-EPO fusion protein following IV injection were investigated in a permanent middle cerebral artery occlusion (MCAO) model in the adult mouse. Following MCAO, mice were treated IV with low (1000 U/kg) and high (10,000 U/kg) doses of recombinant EPO, or with low (0.05 mg/kg) or high (1.0 mg/kg) doses of the cTfRMAb-EPO fusion protein. Hemispheric stroke volume and neural deficit scores were quantitated 24h after MCAO. There was no reduction in stroke volume or neural deficit following the IV administration of either dose of EPO or the low dose of cTfRMAb-EPO fusion protein. However, after treatment with the 1.0 mg/kg dose of the cTfRMAb-EPO fusion protein, the hemispheric stroke volume was reduced 81% and the neural deficit was reduced 78%. These studies demonstrate high degrees of neuroprotection in stroke with EPO when the neurotrophin is re-engineered as an IgG-EPO fusion protein to enable transport across the BBB following IV administration.


Assuntos
Eritropoetina/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Anticorpos Monoclonais , Eritropoetina/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/uso terapêutico , Receptores da Transferrina/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico
10.
J Autoimmun ; 34(2): 145-54, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19796917

RESUMO

The ectoenzyme ADP-ribosyltransferase 2.2 (ART2.2) can apoptotically delete various T-cell subsets. Depending on the involved apoptotic T-cell subset, enhanced ART2.2 activity could result in immunosuppression or autoimmunity. Diminished activity of the CD38 ectoenzyme that normally represents a counter-regulatory competitor for the NAD substrate represents one mechanism enhancing ART2.2 activity. Hence, it would be desirable to develop an agent that efficiently blocks ART2.2 activity in vivo. While the llama derived recombinant s+16 single domain antibody overcame the difficulty of specifically targeting the ART2.2 catalytic site potential therapeutic use of this reagent is limited due to short in vivo persistence. Thus, we tested if a modified version of s+16 incorporating the murine IgG1 Fc tail (s+16Fc) mediated long-term efficient in vivo suppression of ART2.2. We reasoned an ideal model to test the s+16Fc reagent were NOD mice in which genetic ablation of CD38 results in an ART2.2 mediated reduction in already sub-normal numbers of immunoregulatory natural killer T-(NKT) cells to a level that no longer allows them when activated by the super-agonist alpha-galactosylceramide (alpha-GalCer) to elicit effects inhibiting autoimmune type 1 diabetes (T1D) development. Treatment with s+16Fc efficiently mediated long-term in vivo inhibition of ART2.2 activity in NOD.CD38(null) mice, restoring their iNKT cell numbers to levels that upon alpha-GalCer activation were capable of inhibiting T1D development.


Assuntos
ADP Ribose Transferases/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Células T Matadoras Naturais/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , ADP Ribose Transferases/genética , ADP Ribose Transferases/imunologia , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Animais , Diabetes Mellitus Tipo 1/terapia , Feminino , Galactosilceramidas/imunologia , Terapia de Imunossupressão , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Depleção Linfocítica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologia , Engenharia de Proteínas , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia
11.
Mol Immunol ; 45(4): 1027-34, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17804071

RESUMO

Anti-idiotypic antibody (Ab2) 3H6 is directed against the human monoclonal antibody 2F5, which is one of a few neutralising antibodies against HIV-1. Since the binding epitope of 2F5 is cryptic and no neutralising immune response could be elicited by several potential vaccines comprising this region, Ab2/3H6 represents a potent vaccine candidate for active immunisation. Here we describe the molecular features of Ab2/3H6 after changing the antigen binding specificity by single point mutations in the complementarity-determining region 3 of the Ab2/3H6 heavy chain. The resulting Ab2/3H6 mutants were compared in several experimental settings to the wild type Ab2/3H6 Fab fragment. Moreover, we report about an immunisation study with Ab2/3H6 Fab variants, which elicited a specific 2F5-like humoral immune response in BALB/c mice.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/genética , Formação de Anticorpos , Células CHO , Regiões Determinantes de Complementaridade/administração & dosagem , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Cricetinae , Cricetulus , Epitopos , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual
12.
Infect Immun ; 74(2): 1196-203, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16428769

RESUMO

Staphylococcal protein A (SpA) is representative of a new class of antigens, the B-cell superantigens (SAgs). These antigens bind to the Fab regions of immunoglobulin molecules outside their complementarity-determining regions. SpA, the best-studied B-cell SAg, reacts with the Fabs of most VH3+ immunoglobulins, which are expressed on 30 to 60% of human peripheral B cells. Therefore, B-cell SAgs like SpA have great potential to elicit inflammatory responses in vivo. We previously reported that the interaction of SpA with VH3+ immunoglobulin molecules leads to activation of the complement cascade and produces a histologic pattern of inflammation in the skin of a rabbit indicative of immune complex injury. To elucidate the cellular and molecular events contributing to this type of unconventional immune complex-mediated inflammation, we established a mouse peritoneal Arthus reaction model. Mice treated intravenously with human polyclonal immunoglobulin G (IgG), followed by intraperitoneal injection of SpA, showed neutrophil influx into the peritoneal cavity with peak numbers appearing at 8 h. This inflammatory reaction was dependent on the interaction of SpA with VH3+ IgG. Mast cells, FcgammaRIII, complement components, and tumor necrosis factor alpha play obligatory roles, and the reaction is associated with the local release of the CXC chemokines macrophage inflammatory protein 2 and KC. The data provide further compelling evidence for the induction of immune complex-mediated injury by a B-cell SAg and highlight important factors contributing to the pathogenesis of this novel type of inflammatory reaction.


Assuntos
Reação de Arthus/imunologia , Reação de Arthus/fisiopatologia , Linfócitos B/imunologia , Imunoglobulina G/administração & dosagem , Proteína Estafilocócica A/administração & dosagem , Superantígenos/administração & dosagem , Animais , Reação de Arthus/etiologia , Linfócitos B/metabolismo , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/administração & dosagem , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos , Neutrófilos/imunologia , Cavidade Peritoneal/fisiopatologia , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismo , Superantígenos/imunologia , Superantígenos/metabolismo
13.
J Immunol ; 175(4): 2184-90, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16081785

RESUMO

The BCR V region has been implicated as a potential avenue of T cell help for autoreactive B cells in systemic lupus erythematosus. In principle, either germline-encoded or somatically generated sequences could function as targets of such help. Preceding studies have indicated that class II MHC-restricted T cells in normal mice attain a state tolerance to germline-encoded Ab diversity. In this study, we tested whether this tolerance is intact in systemic lupus erythematosus-prone (New Zealand Black x SWR)F1 mice (SNF1). Using a hybridoma sampling approach, we found that SNF1 T cells were tolerant to germline-encoded Ab sequences. Specifically, they were tolerant to germline-encoded sequences derived from a lupus anti-chromatin Ab that arose spontaneously in this strain. This was true both for diseased and prediseased mice. Thus, there does not appear to be a global defect in T cell tolerance to Ab V regions in this autoimmune-prone strain either before or during autoimmune disease.


Assuntos
Diversidade de Anticorpos/genética , Autoanticorpos/genética , Anergia Clonal/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/imunologia , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/biossíntese , Cromatina/imunologia , Feminino , Mutação em Linhagem Germinativa/genética , Hibridomas , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Dados de Sequência Molecular , Hipermutação Somática de Imunoglobulina
14.
J Rheumatol ; 30(4): 680-90, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12672184

RESUMO

OBJECTIVE: To determine the optimal dose regimen for intravenous Ro 45-2081 (lenercept) in patients with rheumatoid arthritis (RA) by evaluating efficacy, safety, tolerability, and pharmacokinetic and pharmacodynamic characteristics. METHODS: Adult patients with longstanding RA who were taking stable doses of nonsteroidal antiinflammatory drug and/or low dose corticosteroids but who had stopped their previous disease-modifying antirheumatic drug were randomly assigned to receive 3 intravenous infusions, one every 4 weeks, of placebo or Ro 45-2081 in a double blind, placebo controlled, parallel group multicenter trial. Patients received one of the following: (1) placebo, (2) low dose Ro 45-2081 (0.05 mg/kg, maximum 5 mg), (3) middle dose (mid-dose) Ro 45-2081 (0.2 mg/kg, maximum 20 mg), or (4) high dose Ro 45-2081 (0.5 mg/kg, maximum 50 mg). Efficacy measures included change from baseline in number of swollen joints and tender joints, scores on physician and patient assessments of disease activity, and patient assessment of pain, as well as acute phase reactants. RESULTS: Patients treated with Ro 45-2081 exhibited improvement after one day of the first intravenous infusion. This treatment benefit maximized by 2 weeks but diminished thereafter. After the second and third infusion, improvement was of shorter duration as non-neutralizing anti-Ro 45-2081 antibodies developed and accelerated clearance of Ro 45-2081. There were no antibodies after the first infusion. This made efficacy transient in the mid-dose group and modest in the low and high dose groups at 12 weeks of treatment, resulting in no statistical differences at most time points or doses of Ro 45-2081. The majority of adverse experiences were mild or moderate, and were not related or only remotely related to study drug. No clinically relevant changes in mean laboratory values were reported. The third dose pharmacokinetic measurements showed that the average Ro 45-2081 clearance rate more than doubled compared with the first dosing interval, thus reducing the average Ro 45-2081 AUC by 36%. CONCLUSION: Intravenous Ro 45-2081 every 4 weeks proved to be well tolerated and transiently effective in the mid-dose group and modestly effective in the low and high dose groups in patients with longstanding RA. The interactions between Ro 45-2081, its non-neutralizing anti-Ro 45-2081 antibody, and the clinical benefit remain complex, but affected efficacy over the 12 weeks of treatment as Ro 45-2081 concentrations fell.


Assuntos
Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Receptores do Fator de Necrose Tumoral/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Adulto , Idoso , Anticorpos/sangue , Antirreumáticos/efeitos adversos , Antirreumáticos/imunologia , Antirreumáticos/farmacocinética , Artrite Reumatoide/imunologia , Método Duplo-Cego , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/efeitos adversos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias gama de Imunoglobulina , Infecções/diagnóstico , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Placebos , Receptores do Fator de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Resultado do Tratamento
15.
J Immunol ; 169(8): 4113-23, 2002 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12370339

RESUMO

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.


Assuntos
Subpopulações de Linfócitos B/patologia , Proteínas do Sistema Complemento/fisiologia , Dermatite de Contato/imunologia , Imunoglobulina M/metabolismo , Cadeias kappa de Imunoglobulina/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Sítios de Ligação de Anticorpos/genética , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Dermatite de Contato/genética , Dimerização , Orelha Externa/imunologia , Edema/genética , Edema/imunologia , Feminino , Haptenos/metabolismo , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/farmacologia , Imunoglobulina M/administração & dosagem , Cadeias kappa de Imunoglobulina/administração & dosagem , Cadeias kappa de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/farmacologia , Injeções Intravenosas , Linfopenia/genética , Linfopenia/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Camundongos Mutantes , Subunidades Proteicas , Trinitrobenzenos/imunologia
16.
J Immunol ; 162(8): 4406-16, 1999 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10201976

RESUMO

Lupus-prone, anti-DNA, heavy (H) chain "knock-in" mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2-3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6-7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vkappa utilization, different from that of nonautoimmune (C57BL/6 x BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vkappa-Jkappa rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.


Assuntos
Anticorpos Antinucleares/biossíntese , Afinidade de Anticorpos/genética , DNA/imunologia , Marcação de Genes , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Lúpus Vulgar/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/química , Anticorpos Antinucleares/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Sequência de Bases , Cruzamentos Genéticos , Feminino , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/administração & dosagem , Região Variável de Imunoglobulina/genética , Lúpus Vulgar/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Transgênicos , Dados de Sequência Molecular
17.
J Autoimmun ; 11(5): 539-46, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9802941

RESUMO

A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305 kDa goat antibody-enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.


Assuntos
Anticorpos Antinucleares/administração & dosagem , Anticorpos Antinucleares/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Sistemas de Liberação de Medicamentos , Animais , Anticorpos Antinucleares/genética , Anticorpos Monoclonais/genética , Sequência de Bases , Células CHO , Células COS , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Cricetinae , Primers do DNA/genética , Proteínas de Fluorescência Verde , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Proteínas Luminescentes/imunologia , Camundongos , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico
18.
Br J Cancer ; 77(9): 1405-12, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9652755

RESUMO

Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue (liver, 2.8 %ID g(-1); lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.


Assuntos
Antígenos de Neoplasias/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Imunoterapia/métodos , Imunotoxinas/metabolismo , Receptor ErbB-2/imunologia , Animais , Afinidade de Anticorpos , Dimerização , Humanos , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Leves de Imunoglobulina/administração & dosagem , Imunotoxinas/administração & dosagem , Camundongos , Camundongos SCID , Distribuição Tecidual
20.
Int J Cancer ; 62(3): 351-5, 1995 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-7628878

RESUMO

B1 (dsFv)-PE38 and B3(dsFv)-PE38 are recombinant immunotoxins in which the Fv fragments of MAbs B1 and B3, respectively, are stabilized by an engineered interchain disulfide bond and are fused at their C-termini to a modified Pseudomonas exotoxin from which the cell-binding domain has been deleted (PE38). Both immunotoxins have been shown to be specifically cytotoxic toward human cancer cell lines which express Le gamma-related carbohydrates on their surface, and when given i.v., eradicated 30- to 50-mm3 s.c. A431 tumors growing in nude mice. A major advantage of dsFv-immunotoxins is their stability at 37 degrees C compared with the relatively unstable single-chain Fvs. This allows them to be given continuously by osmotic pumps placed in the peritoneal cavity. In an attempt to increase the therapeutic index of the immunotoxins, we have now delivered them continuously for 6 days through mini-osmotic pumps placed in the peritoneal cavity of tumor-bearing nude mice. Using this mode of administration, we were able to maintain a constant level of immunotoxin in the serum which was non-toxic to the mice, but caused complete regressions of large 150- to 200-mm3 tumors which lasted for over a month at 1/11 of the LD50 with B1(dsFv)-PE38 and 1/6 of the LD50 with B3(dsFv)-PE38. Complete regression of tumors of similar size could also be achieved by i.v. bolus injections of these immunotoxins at 1/7 of the LD50 with B1(dsFv)-PE38) and 1/3 of the LD50 with B3(dsFv)-PE38. These results suggest that in patients it may be advantageous to administer dsFv-immunotoxins by continuous infusion, since a larger therapeutic index is achieved.


Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Dissulfetos/administração & dosagem , Exotoxinas/administração & dosagem , Imunotoxinas/administração & dosagem , Neoplasias Experimentais/terapia , Fatores de Virulência , Animais , Anticorpos Monoclonais/administração & dosagem , Dissulfetos/química , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Exotoxinas/sangue , Exotoxinas/química , Feminino , Humanos , Fragmentos de Imunoglobulinas/administração & dosagem , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/administração & dosagem , Cadeias Leves de Imunoglobulina/administração & dosagem , Região Variável de Imunoglobulina/administração & dosagem , Imunotoxinas/sangue , Imunotoxinas/química , Bombas de Infusão Implantáveis , Infusões Parenterais , Injeções Intravenosas , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo , Exotoxina A de Pseudomonas aeruginosa
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