RESUMEN
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.
Asunto(s)
Región Variable de Inmunoglobulina/metabolismo , Microbiología Industrial/métodos , Metanol/metabolismo , Pichia/metabolismo , División Celular , Medios de Cultivo , Fermentación , Fibronectinas/inmunología , Región Variable de Inmunoglobulina/genética , Modelos Biológicos , Modelos Teóricos , Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Antígenos de Neoplasias , Biomarcadores de Tumor , Sustancias de Crecimiento/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Serina Endopeptidasas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Secuencia de Bases , Biotecnología , Complejo CD3 , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , ADN Recombinante/genética , Endopeptidasas , Gelatinasas , Vectores Genéticos , Humanos , Inmunoterapia , Técnicas In Vitro , Activación de Linfocitos , Proteínas de la Membrana , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Reacciones Cruzadas , Cartilla de ADN/genética , Endopeptidasas , Gelatinasas , Humanos , Inmunización , Proteínas de la Membrana , Ratones , Ratones Noqueados , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias/genética , Neoplasias/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Especificidad de la Especie , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Antígenos de Superficie/inmunología , Glicoproteínas de Membrana/farmacología , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Especificidad de Anticuerpos , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Células COS , Chlorocebus aethiops , Diseño de Fármacos , Células HeLa/efectos de los fármacos , Humanos , Fragmentos Fab de Inmunoglobulinas , Células Jurkat/efectos de los fármacos , Células KB/efectos de los fármacos , Ligandos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Proteínas de la Membrana/farmacología , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/fisiología , Estructura Terciaria de Proteína , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/fisiología , Solubilidad , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/químicaRESUMEN
The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP(+) stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcinomas. Importantly, compared with the CDR-grafted humanized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Biomarcadores de Tumor , Carcinoma/inmunología , Sustancias de Crecimiento/inmunología , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos de Neoplasias/metabolismo , Carcinoma/metabolismo , Endopeptidasas , Epítopos/inmunología , Proteínas de la Matriz Extracelular/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Gelatinasas , Sustancias de Crecimiento/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo , TemperaturaRESUMEN
Four completely human antibody derivatives [single-chain-antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding-competition studies and analysis by fluorescence-activated cell sorting showed high-affinity binding (10--20 nM) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable for in vivo diagnostic and tumor-targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias , Biomarcadores de Tumor , Sustancias de Crecimiento/inmunología , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Secuencia de Bases , Unión Competitiva , Cartilla de ADN , ADN Complementario , Endopeptidasas , Gelatinasas , Sustancias de Crecimiento/química , Humanos , Inmunohistoquímica , Proteínas de la Membrana , Datos de Secuencia Molecular , Neoplasias/inmunología , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/químicaRESUMEN
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.
Asunto(s)
Anticuerpos/metabolismo , Reacciones Antígeno-Anticuerpo , Neoplasias/inmunología , Tromboplastina/metabolismo , Anticuerpos/inmunología , Secuencia de Bases , Cartilla de ADN , Tromboplastina/químicaRESUMEN
Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.
Asunto(s)
Apoptosis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Agregación de Receptores , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Anticuerpos Monoclonales/inmunología , Proteínas Reguladoras de la Apoptosis , Activación Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/agonistas , Proteína Estafilocócica A/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The interaction of simian foamy viruses (FVs) with their putative cellular receptor(s) was studied with two types of recombinant envelope protein (Env). Transient expression of full-length Env in BHK-21 cells induced syncytia formation. However, selected stable transfectants fused with naive cells but not with each other. A soluble fusion protein of the Env surface domain with the Fc fragment of a human IgG1 heavy chain (EnvSU-Ig) was produced in the baculovirus expression system, purified to homogeneity, and used for binding and competition analyses. EnvSU-Ig but not unrelated Ig fusion proteins bound to cells specifically. Neutralizing serum blocked binding of EnvSU-Ig and, vice versa, serum-mediated neutralization was abrogated by the chimeric protein. Concomitant reduction of EnvSU-Ig binding and FV susceptibility was seen in Env-expressing target cells. Although EnvSU-Ig did not inhibit FV infection, very likely due to its displacement by multivalent virus-cell interactions, this divalent ligand should help to characterize functionally and to identify the ubiquitous FV receptor.
Asunto(s)
Glicoproteínas/metabolismo , Spumavirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Cricetinae , Expresión Génica , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Pruebas de Neutralización , Pan troglodytes , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Spodoptera , Spumavirus/genética , Spumavirus/fisiología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/genética , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Biblioteca de Péptidos , Proteínas Virales de Fusión/genética , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/aislamiento & purificación , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Bacteriófagos , Baculoviridae/genética , Proteínas de la Cápside , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Vectores Genéticos , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera/citología , Técnica de Sustracción , Toxoide Tetánico/inmunologíaRESUMEN
Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Región Variable de Inmunoglobulina/biosíntesis , Proteus mirabilis/genética , Animales , Complejo Antígeno-Anticuerpo , Antígenos CD/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/crecimiento & desarrollo , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Proteus mirabilis/citología , Proteus mirabilis/crecimiento & desarrollo , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos/genética , Clonación Molecular/métodos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Receptores de Interferón/inmunología , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/aislamiento & purificación , Afinidad de Anticuerpos/genética , Genes de Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Receptor de Interferón gammaRESUMEN
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.
Asunto(s)
Baculoviridae/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Spodoptera/virología , Animales , Células COS/fisiología , Línea Celular Transformada , Clonación Molecular , Cricetinae , Técnicas de Transferencia de Gen , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/farmacología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/farmacología , Riñón/fisiología , Ratones , SolubilidadRESUMEN
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.
Asunto(s)
Colagenasas/metabolismo , Glicoproteínas/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Células Cultivadas , Colagenasas/genética , Activación Enzimática , Glicoproteínas/genética , Humanos , Metaloproteinasa 1 de la Matriz , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Spodoptera/enzimología , Spodoptera/genética , Inhibidores Tisulares de MetaloproteinasasRESUMEN
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.
Asunto(s)
Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Citotoxicidad Inmunológica , Humanos , Ligandos , Mutación , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Solubilidad , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antígenos CD/química , Inmunoadhesinas CD4/farmacología , Interferón gamma/antagonistas & inhibidores , Estructura Terciaria de Proteína , Receptores de Interferón/química , Animales , Afinidad de Anticuerpos , Antivirales/farmacología , Inmunoadhesinas CD4/biosíntesis , Inmunoadhesinas CD4/genética , Línea Celular , Humanos , Insectos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Solubilidad , Especificidad de la Especie , Receptor de Interferón gammaRESUMEN
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.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Animales , Western Blotting , Células Cultivadas , Quimotripsina/metabolismo , Cartilla de ADN , Expresión Génica , Proteasa del VIH/metabolismo , Transcriptasa Inversa del VIH , Humanos , Insectos , Cinética , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Inmunológica , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Antígenos CD4/genética , VIH-1/fisiología , Biosíntesis de Proteínas , Replicación Viral/genética , Línea Celular , HumanosRESUMEN
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.