RESUMO
TNF-related apoptosis-inducing ligand (TRAIL) is a typical member of the tumor necrosis factor (TNF) ligand family that is expressed as a type II membrane protein (memTRAIL) and signals apoptosis via the death domain-containing receptors TRAIL-R1 and -2. Soluble recombinant derivatives of TRAIL (sTRAIL) are considered as novel tumors therapeutics because of their selective apoptosis inducing activity in a variety of human tumors but not in normal cells. Using antagonistic antigen-binding fragment (Fab) preparations of TRAIL-R1- and TRAIL-R2-specific antibodies, we demonstrate in this study that TRAIL-R1 becomes activated by both the soluble and the membrane-bound form of the ligand, whereas TRAIL-R2 becomes only activated by memTRAIL or soluble TRAIL secondarily cross-linked by antibodies. Furthermore, we show that the restricted signal capacity of sTRAIL can be readily converted into a fully signal competent memTRAIL-like molecule, i.e. a TRAIL-R2 stimulating ligand, by genetic fusion to an antibody derivative that allows antigen-dependent 'immobilization' of the fusion protein to cell surfaces. We conclude that antibody targeting-dependent activation can be used to design selective therapeutics derived of those ligands of the TNF family that are biologically inactive in their soluble form.
Assuntos
Antígenos de Superfície/imunologia , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Especificidade de Anticorpos , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Células COS , Chlorocebus aethiops , Desenho de Fármacos , Células HeLa/efeitos dos fármacos , Humanos , Fragmentos Fab das Imunoglobulinas , Células Jurkat/efeitos dos fármacos , Células KB/efeitos dos fármacos , Ligantes , Glicoproteínas de Membrana/química , Proteínas de Membrana/química , Proteínas de Membrana/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/fisiologia , Estrutura Terciária de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/fisiologia , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/químicaRESUMO
Different soluble human TNFR80 derivatives, a solubilized form of the complete TNFR80, the TNFR80 extracellular domain, a secretory TNFR80 mutant (TR80TM-) with a deleted transmembrane region, and a TNFR80 immunoadhesin were produced in insect cells and characterized side by side with a recombinant human TNFR60 extracellular domain with respect to TNF binding affinity and neutralization of TNF bioactivity. The construct TR80TM- and the solubilized complete TNFR80 revealed a similar TNF binding and neutralization capacity, which was superior to the monovalent TNFR80 extracellular domain and comparable to the bivalent TNFR80 immunoadhesin, already known as a potent TNF antagonist. Determination of ligand off rate constants of the various receptor constructs by surface plasmon resonance revealed a correlation of low off rates with a high TNF neutralization capacity. We propose that the high TNF binding and neutralization capacity of the solubilized complete TNFR80 and TR80TM- in comparison with the monovalent extracellular TNR80 domain is due to a noncovalent self-aggregation of the receptors via their intracellular domain. This finding suggests that efficient soluble TNF antagonists can be derived from TNFR themselves without the need of construction of TNFR Ig Fc fusion proteins.
Assuntos
Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Citotoxicidade Imunológica , Humanos , Ligantes , Mutação , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
We describe here the bioengineering of a bivalent IFN-gamma-RFc immunoadhesin consisting of the extracellular domain of the human IFN-gamma receptor alpha chain (IFN-gamma-R) fused to a human IgG1 Fc region (encoding hinge, CH2 and CH3 domain) that was efficiently expressed as a covalently linked homodimer in insect cells and purified in a one-step purification procedure. The IFN-gamma-RFc fusion protein exerted a 3-fold higher ligand binding affinity in binding competition studies in vitro compared with the monovalent extracellular IFN-gamma-R domain. In addition, the in vitro antagonistic activity of IFN-gamma-RFc, as determined by inhibition of IFN-gamma-induced virus protection and HLA-DR expression, was more than 30-fold higher in comparison with the monovalent soluble receptor. The described IFN-gamma-R immunoadhesin is a potential therapeutic reagent to interfere with the disease-promoting activities of IFN-gamma in several autoimmune diseases.
Assuntos
Antígenos CD/química , Imunoadesinas CD4/farmacologia , Interferon gama/antagonistas & inibidores , Estrutura Terciária de Proteína , Receptores de Interferon/química , Animais , Afinidade de Anticorpos , Antivirais/farmacologia , Imunoadesinas CD4/biossíntese , Imunoadesinas CD4/genética , Linhagem Celular , Humanos , Insetos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Solubilidade , Especificidade da Espécie , Receptor de Interferon gamaRESUMO
We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.
Assuntos
Proteínas de Ligação a DNA/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/isolamento & purificação , Biblioteca de Peptídeos , Proteínas Virais de Fusão/genética , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Bacteriófagos , Baculoviridae/genética , Proteínas do Capsídeo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Vetores Genéticos , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera/citologia , Técnica de Subtração , Toxoide Tetânico/imunologiaRESUMO
The products of the endogenous reverse transcriptase reaction of SIVagmTYO-7 were characterized after the reaction conditions had been optimized. The major reaction product in the presence of actinomycin D and oligo(dT) was a DNA with a size of 300 bases. Without actinomycin D two additional reaction products with 600 or 700 bases appeared. The 300 base product was identified as the (-)strong-stop DNA, whereas the 700 base product is the (+)strong-stop DNA. The 600 base product appeared only after oligo(dT) priming. The (-)strong-stop DNA hybridized specifically with a 9 kb RNA found in virus particles and three RNA species of 1.8, 4.8 and 9 kb isolated from SIVagmTYO-7 infected cells.
Assuntos
DNA Polimerase Dirigida por RNA/isolamento & purificação , Vírus da Imunodeficiência Símia/enzimologia , Animais , Northern Blotting , Linhagem Celular , Chlorocebus aethiops , Sondas de Oligonucleotídeos , RNA Viral/química , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificaçãoRESUMO
The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.
Assuntos
Produtos do Gene gag/biossíntese , Produtos do Gene pol/biossíntese , HIV-2/genética , Sequência de Bases , DNA Recombinante/biossíntese , DNA Recombinante/genética , Produtos do Gene gag/genética , Produtos do Gene gag/ultraestrutura , Produtos do Gene pol/genética , Produtos do Gene pol/ultraestrutura , Genes gag/fisiologia , Genes pol/fisiologia , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/fisiologia , HIV-2/química , HIV-2/ultraestrutura , Dados de Sequência Molecular , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/fisiologia , Processamento de Proteína Pós-TraducionalRESUMO
Bispecific antibodies directed against tumour associated antigens and the T cell receptor component CD3 for recruitment and tumour targeted activation of T cells represent a novel class of highly specific immunotherapeutics for cancer. We here describe the construction, eukaryotic expression and in vitro functional activity of a new T cell activating bispecific reagent, termed TTS for T cell targeting to the tumour stroma, comprised of a CD3 specific single chain antibody derivative (scFv) fused C-terminally to a 'fibroblast activation protein' (FAP) specific scFv that targets cytotoxic effector cells to FAP. FAP is highly expressed in the vascularised tumoural stroma of most lung, breast and colon carcinomas. It thus represents a selectively tumour associated, yet common marker of many solid tumours and is a potentially ideal candidate marker for efficient targeting of immune effector cells.
Assuntos
Anticorpos Biespecíficos/biossíntese , Antígenos de Neoplasias , Biomarcadores Tumorais , Substâncias de Crescimento/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Serina Endopeptidases/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Sequência de Bases , Biotecnologia , Complexo CD3 , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , DNA Recombinante/genética , Endopeptidases , Gelatinases , Vetores Genéticos , Humanos , Imunoterapia , Técnicas In Vitro , Ativação Linfocitária , Proteínas de Membrana , Células Tumorais CultivadasRESUMO
OBJECTIVE: The guided selection strategy for isolation of human antibody (Ab) fragments specific for human interferon gamma receptor 1 (IFNGR-1) from a cloned Ab VH and VL repertoire has been investigated. In order to identify recombinant Abs binding to soluble antigen, a novel method termed affinity sedimentation was introduced here. RESULTS AND CONCLUSIONS: The VH region of murine monoclonal Ab (IR gamma-1) against human IFNGR-1 was combined with human VL repertoire and used for selection of human VL regions. One of these human VL regions (kappa 2) possesses high homology to the murine template VL region, also in CDR3 (77%). A chimeric Fab consisting of kappa 2 and the murine IR gamma-1 VH region was highly IFNGR-1 specific and exerted the same epitope specificity and a comparable binding affinity as the parental murine Fab. In a further step, the selected human VL region kappa 2 was combined with a human VH repertoire and led by guided selection to the generation of a completely human Fab (1b5) specific for human IFNGR-1. The overall VH region homology of 1b5 compared to the parental antibody IR gamma-1 was 81%, with a rather low homology in CDR3. Binding competition studies revealed that the epitope recognized by 1b5 differs from the parental Ab IR gamma-1.
Assuntos
Anticorpos/imunologia , Especificidade de Anticorpos/genética , Clonagem Molecular/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Receptores de Interferon/imunologia , Sequência de Aminoácidos , Anticorpos/genética , Anticorpos/isolamento & purificação , Afinidade de Anticorpos/genética , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Receptor de Interferon gamaRESUMO
An adherent human cell line (293) was made susceptible for HIV-1 infection by transfer of a CD4 expression plasmid. These cells could be infected with HIV-1 and produced infectious virus up to a titer of 10(6) TCID50/ml releasing p24 protein up to 1 micrograms/ml. Since they can be efficiently transfected with reporter genes, these cells are a suitable model system to monitor biochemical events during productive infection of HIV-1 and can be used for antiviral drugs. Translational frameshifting determines the balance of the structural Gag versus the catalytic Pol proteins which is probably crucial for correct virus assembly. We have genetically engineered CD4 expressing 293 cells with a sensitive in vivo reporter system to monitor the extent of frameshifting in HIV-1-infected versus uninfected cells. During the time course of productive HIV-1 infection the low efficiency of ribosomal frameshifting is not altered.
Assuntos
Antígenos CD4/genética , HIV-1/fisiologia , Biossíntese de Proteínas , Replicação Viral/genética , Linhagem Celular , HumanosRESUMO
We here describe an efficient procedure for overexpression and purification of recombinant complete human interferon-gamma (IFN-gamma) receptor (IFN-gamma-R) and its extracellular fragment employing a baculovirus (BV) expression system. Infection of Sf 158 cells with recombinant BV results in membrane expression of high affinity IFN-gamma-R (Kd 1.6 x 10(-10) M), with approximately 10(6) molecules/cell 40 h postinfection. Solubilized, affinity-purified IFN-gamma-R and a secreted extracellular domain of IFN-gamma-R were compared for ligand-binding capacity and antagonistic activity in an IFN-gamma bioassay. Our results show that the complete receptor has a 2.5-fold higher ligand affinity and a 15-fold higher IFN-gamma in vitro-neutralizing capacity in an in vitro virus protection assay as compared to the extracellular fragment. This suggests that the transmembrane and cytoplasmic domains of IFN-gamma-R contribute to stability and/or enhance formation of biologically active receptor complexes in solution.
Assuntos
Receptores de Interferon/genética , Animais , Baculoviridae , Sequência de Bases , Cromatografia de Afinidade , Primers do DNA/genética , Expressão Gênica , Vetores Genéticos , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Dados de Sequência Molecular , Mariposas , Testes de Neutralização , Receptores de Interferon/isolamento & purificação , Receptores de Interferon/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Receptor de Interferon gamaRESUMO
For investigation of a possible physical interaction between the two human tumor necrosis factor receptors, TR60 (type I) and TR80 (type II), the baculovirus expression system was used. Each of the receptors was expressed as a membrane-integrated protein in insect cells, able to specifically bind the two ligands, tumor necrosis factor (TNF) and lymphotoxin (LT alpha). Typically, about 150,000 membrane receptors per cell could be detected 40 h after infection, exerting high affinity ligand binding capacity with Kd values virtually identical to that of human cell lines. The baculovirus system allowed coexpression of both TNF membrane receptors at very high and about equal numbers to investigate the existence of heteromultimeric receptor complexes, either formed spontaneously or ligand induced. Neither saturation binding studies nor immunoprecipitation experiments gave an indication for the existence of TNF receptor heteromers. These data are in accordance with the current view of TNF signaling, in which homonultimerization, rather than heteromer formation of TNF receptors is the initial activating event.
Assuntos
Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Linhagem Celular , Expressão Gênica , Humanos , Cinética , Linfotoxina-alfa/metabolismo , Nucleopoliedrovírus/genética , Receptores do Fator de Necrose Tumoral/classificação , Spodoptera , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.
Assuntos
Cercopithecus/microbiologia , Chlorocebus aethiops/microbiologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Imunodeficiência Símia/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Cinética , Peso Molecular , Polinucleotídeos , DNA Polimerase Dirigida por RNA/isolamento & purificação , Vírus da Imunodeficiência Símia/isolamento & purificação , Relação Estrutura-Atividade , Moldes GenéticosRESUMO
Solid tumours growing beyond a size of 1-2 mm in diameter induce supporting connective tissue structures, the tumour stroma, comprising activated fibroblasts and newly formed blood vessels, embedded in an extracellular matrix. The selective destruction of this tissue or the inhibition of its function (e.g. tumour neoangiogenesis) may result in the destruction of tumour nodules, thus providing novel opportunities for tumour therapy. Our approach aims at an antibody-mediated induction of coagulation in tumour nodules to cut off their blood supply. As a target structure the fibroblast activation protein (FAP) is used, which is specifically and abundantly expressed on the activated fibroblasts of the tumour stroma. We constructed a fusion protein comprising a single-chain module of a FAP-specific humanized antibody [single-chain fragment variable (scFv) OS4] and the extracellular domain of human tissue factor. The fusion protein, designated TFOS4, was produced in the Proteus mirabilis protoplast expression system with a yield of 15 microg/ml. Biochemical characterization of TFOS4 revealed high-affinity binding to cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted allosteric activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to FAP-expressing cells efficiently generated activated factor X. Finally, cell-bound TFOS4 selectively induced plasma coagulation, implying its activity under physiological conditions, notably with relevant concentrations of coagulation factors and their natural inhibitors. These findings suggest that TFOS4 has the potential to increase the procoagulant state in a cell-type-specific fashion. No systemic coagulation or side effects were observed when TFOS4 was injected intravenously into normal mice, indicating the biosafety and specificity of the recombinant protein.
Assuntos
Anticorpos/metabolismo , Reações Antígeno-Anticorpo , Neoplasias/imunologia , Tromboplastina/metabolismo , Anticorpos/imunologia , Sequência de Bases , Primers do DNA , Tromboplastina/químicaRESUMO
Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active-site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)-MMP-1 proteins are able to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1.
Assuntos
Colagenases/metabolismo , Glicoproteínas/metabolismo , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Células Cultivadas , Colagenases/genética , Ativação Enzimática , Glicoproteínas/genética , Humanos , Metaloproteinase 1 da Matriz , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Spodoptera/enzimologia , Spodoptera/genética , Inibidores Teciduais de MetaloproteinasesRESUMO
To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.
Assuntos
Baculoviridae/genética , Vetores Genéticos , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Western Blotting , Células Cultivadas , Quimotripsina/metabolismo , Primers do DNA , Expressão Gênica , Protease de HIV/metabolismo , Transcriptase Reversa do HIV , Humanos , Insetos , Cinética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMO
Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.
Assuntos
Mutação da Fase de Leitura , Produtos do Gene gag/genética , Produtos do Gene pol/genética , HIV-1/genética , Processamento de Proteína Pós-Traducional , Transfecção , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Expressão Gênica , Produtos do Gene gag/metabolismo , Produtos do Gene pol/metabolismo , Protease de HIV/metabolismo , Humanos , Hidrólise , Camundongos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
BACKGROUND: Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity. MATERIAL AND METHODS: An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP. RESULTS: High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model. CONCLUSIONS: MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.
Assuntos
Antígenos de Neoplasias , Substâncias de Crescimento/genética , Substâncias de Crescimento/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Reações Cruzadas , Primers do DNA/genética , Endopeptidases , Gelatinases , Humanos , Imunização , Proteínas de Membrana , Camundongos , Camundongos Knockout , Camundongos Nus , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/imunologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade da Espécie , Transplante HeterólogoRESUMO
Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Imunológica , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Humanos , Camundongos , Dados de Sequência MolecularRESUMO
Single chain antibodies (scFv) are usually produced in E. coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems. We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs. The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases. Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells. In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence. Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively. Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C. We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.
Assuntos
Baculoviridae/genética , Fragmentos de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Spodoptera/virologia , Animais , Células COS/fisiologia , Linhagem Celular Transformada , Clonagem Molecular , Cricetinae , Técnicas de Transferência de Genes , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/farmacologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/farmacologia , Rim/fisiologia , Camundongos , SolubilidadeRESUMO
Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.