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1.
ACS Infect Dis ; 3(7): 479-491, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28591513

RESUMO

Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Anti-HIV/química , Proteína do Núcleo p24 do HIV/química , HIV-1/química , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Sítios de Ligação , Camelídeos Americanos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/isolamento & purificação , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/imunologia , Humanos , Cinética , Simulação de Dinâmica Molecular , Biblioteca de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Eletricidade Estática
2.
Stem Cells Dev ; 25(23): 1808-1817, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27733096

RESUMO

Hypertrophic differentiation occurs during in vitro chondrogenesis of mesenchymal stem cells (MSCs), decreasing the quality of the cartilage construct. Previously we identified WNT pathway antagonists Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in blocking hypertrophic differentiation of human MSCs (hMSCs). In this study, we investigated the role of endogenously expressed DKK1 and FRZB in chondrogenesis of hMSC and chondrocyte redifferentiation and in preventing cell hypertrophy using three relevant human cell based systems, isolated hMSCs, isolated primary human chondrocytes (hChs), and cocultures of hMSCs with hChs for which we specifically designed neutralizing nano-antibodies. We selected and tested variable domain of single chain heavy chain only antibodies (VHH) for their ability to neutralize the function of DKK1 or FRZB. In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan and collagen type II staining were significantly reduced in monocultured hMSCs and monocultured chondrocytes. Furthermore, in cocultures, cells in pellets showed hypertrophic differentiation. In conclusion, endogenous expression of the WNT antagonists DKK1 and FRZB is necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are indispensable for the initial steps of chondrogenic differentiation of hMSCs, second they are necessary for chondrocyte redifferentiation, and finally in preventing hypertrophic differentiation of articular chondrocytes.


Assuntos
Técnicas de Cultura de Células/métodos , Condrócitos/metabolismo , Condrogênese , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Anticorpos Neutralizantes/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Técnicas de Cocultura , Glicoproteínas/imunologia , Humanos , Hipertrofia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Anticorpos de Domínio Único/imunologia , Via de Sinalização Wnt/efeitos dos fármacos
3.
Sci Rep ; 6: 31621, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27546726

RESUMO

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos Imunológicos , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Células A549 , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/farmacologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/farmacologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Imaging Biol ; 18(4): 535-44, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26589824

RESUMO

PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS). RESULTS: Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR. CONCLUSIONS: These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer.


Assuntos
Hipóxia/patologia , Neoplasias Mamárias Animais/patologia , Imagem Molecular/métodos , Imagem Óptica/métodos , Anticorpos de Domínio Único/metabolismo , Animais , Anidrase Carbônica IX/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Hipóxia Celular , Técnicas de Visualização da Superfície Celular , DNA/metabolismo , Feminino , Células HeLa , Humanos , Imunização , Imuno-Histoquímica , Camundongos , Invasividade Neoplásica , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Mol Cancer Ther ; 14(11): 2527-40, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26351325

RESUMO

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.


Assuntos
Anticorpos/farmacologia , Especificidade de Anticorpos/imunologia , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Camelídeos Americanos/imunologia , Neoplasias/tratamento farmacológico , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos/imunologia , Western Blotting , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/imunologia , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/química , Proteína Morfogenética Óssea 4/imunologia , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/imunologia , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Células HT29 , Humanos , Camundongos , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/metabolismo , Ligação Proteica/imunologia , Estrutura Terciária de Proteína
6.
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
7.
Eur J Nucl Med Mol Imaging ; 40(11): 1718-29, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23778558

RESUMO

PURPOSE: Molecular optical imaging using monoclonal antibodies is slow with low tumour to background ratio. We used anti-HER2 VHHs conjugated to IRDye 800CW to investigate their potential as probes for rapid optical molecular imaging of HER2-positive tumours by the determination of tumour accumulation and tumour to background levels. METHODS: Three anti-HER2 VHHs (11A4, 18C3, 22G12) were selected with phage display and produced in Escherichia coli. Binding affinities of these probes to SKBR3 cells were determined before and after site-specific conjugation to IRDye 800CW. To determine the potential of VHH-IR as imaging probes, serial optical imaging studies were carried out using human SKBR3 and human MDA-MB-231 xenograft breast cancer models. Performance of the anti-HER2 VHH-IR was compared to that of trastuzumab-IR and a non-HER2-specific VHH-IR. Image-guided surgery was performed during which SKBR3 tumour was removed under the guidance of the VHH-IR signal. RESULTS: Site-specific conjugation of IRDye 800CW to three anti-HER2 VHHs preserved high affinity binding with the following dissociation constants (KD): 11A4 1.9 ± 0.03, 18C3 14.3 ± 1.8 and 22G12 3.2 ± 0.5 nM. Based upon different criteria such as binding, production yield and tumour accumulation, 11A4 was selected for further studies. Comparison of 11A4-IR with trastuzumab-IR showed ∼20 times faster tumour accumulation of the anti-HER2 VHH, with a much higher contrast between tumour and background tissue (11A4-IR 2.5 ± 0.3, trastuzumab-IR 1.4 ± 0.4, 4 h post-injection). 11A4-IR was demonstrated to be a useful tool in image-guided surgery. CONCLUSION: VHH-IR led to a much faster tumour accumulation with high tumour to background ratios as compared to trastuzumab-IR allowing same-day imaging for clinical investigation as well as image-guided surgery.


Assuntos
Benzenossulfonatos/farmacocinética , Corantes Fluorescentes/farmacocinética , Indóis/farmacocinética , Imagem Óptica/métodos , Receptor ErbB-2/imunologia , Anticorpos de Domínio Único/imunologia , Cirurgia Assistida por Computador/métodos , Animais , Afinidade de Anticorpos , Benzenossulfonatos/uso terapêutico , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Camelídeos Americanos , Corantes Fluorescentes/uso terapêutico , Humanos , Indóis/uso terapêutico , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Anticorpos de Domínio Único/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS Pathog ; 9(3): e1003202, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23505368

RESUMO

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.


Assuntos
Anticorpos Neutralizantes/imunologia , Camelídeos Americanos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunização , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Neutralização , Proteolipídeos/administração & dosagem , Proteolipídeos/imunologia , Anticorpos de Domínio Único , Ressonância de Plasmônio de Superfície
9.
Eur J Pharm Sci ; 42(4): 332-9, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21220006

RESUMO

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.


Assuntos
Isoenzimas/imunologia , Proteína Quinase C-épsilon , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Sequência de Aminoácidos , Animais , Camelídeos Americanos/imunologia , Regiões Determinantes de Complementaridade , Humanos , Cadeias Pesadas de Imunoglobulinas , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Biblioteca de Peptídeos , Ligação Proteica , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/imunologia
10.
Shock ; 34(2): 125-32, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20016410

RESUMO

Staphylococcus aureus produces the superantigen toxic shock syndrome toxin 1 (TSST-1). When the bacterium invades the human circulation, this toxin can induce life-threatening gram-positive sepsis. Current sepsis treatment does not remove bacterial toxins. Variable domains of llama heavy-chain antibodies (VHH) against toxic shock syndrome toxin 1 ([alpha]-TSST-1 VHH) were previously found to be effective in vitro. We hypothesized that removing TSST-1 with [alpha]-TSST-1 VHH hemofiltration filters would ameliorate experimental sepsis in pigs. After assessing in vitro whether timely removing TSST-1 interrupted TSST-1-induced mononuclear cell TNF-[alpha] production, VHH-coated filters were applied in a porcine sepsis model. Clinical course, survival, plasma interferon [gamma], and TSST-1 levels were similar with and without VHH-coated filters as were TSST-1 concentrations before and after the VHH filter. Plasma TSST-1 levels were much lower than anticipated from the distribution of the amount of infused TSST-1, suggesting compartmentalization to space or adhesion to surface not accessible to hemofiltration or pheresis techniques. Removing TSST-1 from plasma was feasible in vitro. However, the [alpha]-TSST-1 VHH adsorption filter-based technique was ineffective in vivo, indicating that improvement of VHH-based hemofiltration is required. Sequestration likely prevented the adequate removal of TSST-1. The latter warrants further investigation of TSST-1 distribution and clearance in vivo.


Assuntos
Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Choque Séptico/prevenção & controle , Animais , Toxinas Bacterianas , Camelídeos Americanos/imunologia , Células Cultivadas , Enterotoxinas , Feminino , Hemofiltração/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Choque Séptico/imunologia , Superantígenos , Sus scrofa
11.
Biotechnol Bioeng ; 104(1): 143-51, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19475676

RESUMO

Toxic-shock syndrome is primarily caused by the Toxic-shock syndrome toxin 1 (TSST-1), which is secreted by the Gram-positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V(beta)-specific T-cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy-chain antibodies (VHH) in the immuno capturing of TSST-1 from plasma. Data is presented that the selected VHHs are highly specific for TSST-1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST-1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST-1 more than 96% of TSST-1 was depleted from pig plasma. These data pave the way to further explore application of high-affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/métodos , Enterotoxinas/isolamento & purificação , Plasma/química , Staphylococcus aureus/patogenicidade , Superantígenos/isolamento & purificação , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Camelídeos Americanos , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Humanos , Ligação Proteica , Sensibilidade e Especificidade , Superantígenos/imunologia
12.
Mol Immunol ; 46(10): 2022-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19359042

RESUMO

Therapeutic approaches aimed at targeting tumor surface markers using monoclonal antibodies provide a powerful strategy in cancer treatment. Here we report selection of single variable domains (VHH) of llama heavy chain antibodies, using a VHH-phage-display library. A reverse proteomic approach was used to identify the cognate proteins recognized by enriched VHH on HeLa cells. One of these VHH bound the integrin alpha 3 beta 1 (VLA-3) and was further characterized. Most interestingly, this VHH could inhibit VLA-3 mediated cell-matrix adhesion. Our approach provides a fast and efficient method to screen for novel cell surface markers on normal and tumor cells that may find diagnostic or therapeutic application in disease management or treatment.


Assuntos
Anticorpos/análise , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Integrina alfa3beta1/imunologia , Proteômica/métodos , Adesividade , Sequência de Aminoácidos , Animais , Camelídeos Americanos , Adesão Celular , Membrana Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/imunologia , Imunofluorescência , Células HeLa , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Imunoprecipitação , Integrina alfa3beta1/química , Queratinócitos/citologia , Queratinócitos/imunologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Reprodutibilidade dos Testes
13.
Biochem J ; 409(1): 193-8, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17910535

RESUMO

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.


Assuntos
Bioquímica/métodos , Lactococcus lactis/metabolismo , Proteínas Recombinantes/química , Triptofano/análogos & derivados , Triptofano/química , Proteínas de Bactérias/química , Clonagem Molecular , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glicina/análogos & derivados , Glicina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glifosato
14.
Clin Vaccine Immunol ; 13(10): 1079-86, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16928888

RESUMO

Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.


Assuntos
Camelídeos Americanos/imunologia , Fragmentos Fab das Imunoglobulinas/fisiologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Transdução de Sinais/imunologia , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva/imunologia , Células Cultivadas , Escherichia coli/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Neisseria meningitidis/imunologia , Ligação Proteica/imunologia
15.
Methods ; 38(2): 144-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16414272

RESUMO

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Imunidade nas Mucosas/imunologia , Lactococcus lactis/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/química , Administração Intranasal , Animais , Formação de Anticorpos/imunologia , Antígenos/química , Antígenos/genética , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Sítios de Ligação/genética , Expressão Gênica/genética , Vetores Genéticos/genética , Temperatura Alta , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lactococcus lactis/química , Lactococcus lactis/genética , Pulmão/imunologia , Camundongos , Muramidase/genética , Nariz/imunologia , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus pneumoniae/imunologia , Transformação Genética , Ácido Tricloroacético/química
16.
J Biol Chem ; 278(26): 23874-81, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12684515

RESUMO

The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.


Assuntos
Parede Celular/química , Bactérias Gram-Positivas/química , Peptidoglicano/química , Bacillus subtilis/química , Bacillus subtilis/ultraestrutura , Sítios de Ligação , Parede Celular/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/ultraestrutura , Bactérias Gram-Positivas/ultraestrutura , Lactobacillus/química , Lactobacillus/ultraestrutura , Lactococcus lactis/química , Lactococcus lactis/ultraestrutura , Muramidase/metabolismo , Peptidoglicano/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Ácido Nucleico , Streptococcus/química , Streptococcus/ultraestrutura
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