RESUMEN
The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS) studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs) showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antineoplásicos/inmunología , Proteínas de Choque Térmico/inmunología , Inmunoglobulina M/inmunología , Chaperón BiP del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Inmovilizadas/inmunología , SolucionesRESUMEN
Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors.
Asunto(s)
Células Asesinas Naturales/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Anticuerpos de Cadena Única/farmacología , Animales , Afinidad de Anticuerpos , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones SCID , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Células Asesinas Naturales/inmunología , Receptores de Células Asesinas Naturales/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (V(L)-0-V(H)) trimeric/tetrameric "multimer" of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab')(2) following radiolabeling with the Indium-111 ((111)In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab')(2) were 73% and 53.2%, and binding affinities (K(a)) were 1.58 x 10(7) M(1) and 4.31 x 10(6) M (1) for the multimer and F(ab')(2), respectively. Maximal tumor uptake in Le(y)-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 +/- 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 +/- 2.1 %ID/g at 24 hours postinjection for the F(ab')(2). However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities.
Asunto(s)
Inmunoconjugados/farmacocinética , Región Variable de Inmunoglobulina/inmunología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Neoplasias/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales Humanizados , Área Bajo la Curva , Línea Celular Tumoral , Cromatografía en Gel , Estabilidad de Medicamentos , Femenino , Humanos , Inmunoconjugados/sangre , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Radioisótopos de Indio , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/diagnóstico por imagen , Neoplasias/inmunología , Cintigrafía , Proteínas Recombinantes/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Distribución Tisular , Trasplante HeterólogoRESUMEN
Antibody engineering and protein design have led to the creation of a new era of targeted anti-inflammatory therapies in rheumatology. Recombinant DNA technologies have enabled the selection and humanization of specific antibody fragments in order to develop therapeutic reagents of any specificity that can be 'armed' to deliver effective anti-inflammatory 'payloads'. Antibodies and antibody-like proteins provide the opportunity to block key soluble mediators of inflammation in their milieu, or alternatively to block intracellular inflammation-triggering pathways by binding to an upstream cell-surface receptor. These designer proteins can be tuned for desired pharmacokinetic and pharmacodynamic effects, and represent tools for specific therapeutic intervention by delivering precisely the required immunosuppressive effect. The extent of desired and undesired effects of a particular biologic therapy, however, can be broader than initially predicted and require careful evaluation during clinical trials. This Review highlights advances in recombinant technologies for the development of novel biologic therapies in rheumatology.
Asunto(s)
Anticuerpos/uso terapéutico , Diseño de Fármacos , Proteínas Recombinantes/uso terapéutico , Enfermedades Reumáticas/tratamiento farmacológico , Reumatología/tendencias , Animales , Anticuerpos/química , Humanos , Estructura Molecular , Ingeniería de Proteínas , Proteínas Recombinantes/químicaRESUMEN
In vitro compartmentalization (IVC) is a powerful tool for studying protein-protein reactions, due to its high capacity and the versatility of droplet technologies. IVC bridges the gap between chemistry and biology as it enables the incorporation of unnatural amino acids with modifications into biological systems, through protein transcription and translation reactions, in a cell-like microdrop environment. The quest for the ultimate chip for protein studies using IVC is the drive for the development of various microfluidic droplet technologies to enable these unusual biochemical reactions to occur. These techniques have been shown to generate precise microdrops with a controlled size. Various chemical and physical phenomena have been utilized for on-chip manipulation to allow the droplets to be generated, fused, and split. Coupled with detection techniques, droplets can be sorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip. With further technological development of the detection module, factors such as addressable storage, transport and interfacing technologies, could be integrated and thus provide platforms for protein studies with high efficiency and accuracy that conventional laboratories cannot achieve.
Asunto(s)
Evolución Molecular Dirigida/instrumentación , Evolución Molecular Dirigida/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Mapeo de Interacción de Proteínas/instrumentación , Mapeo de Interacción de Proteínas/métodos , Evolución Molecular Dirigida/tendencias , Diseño de Equipo , Técnicas Analíticas Microfluídicas/tendencias , Mapeo de Interacción de Proteínas/tendenciasRESUMEN
Novel in vitro methods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated using in vitro cell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.
Asunto(s)
Anticuerpos/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Ribosomas/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Engineered antibodies have become an invaluable source of biopharmaceuticals against a wide range of diseases. About 200 antibody-based biologicals have been tested in clinical trials. Single chain variable fragments of antibodies (scFvs) provide binding specificity and offer an increased ease of in vitro display selection. Here, we present the generation of a human scFv library from peripheral blood lymphocyte RNA of a patient with relapsed T-cell non-Hodgkin lymphoma (T-NHL) who experienced a rare case of "spontaneous" remission. Antibodies against human T-cell antigen CD28, a co-stimulatory protein that influences the immune response by amplification of the transcriptional effects of T-cell receptors, might have contributed to the patient's remission. The scFv library was panned against CD28 using ribosome display and further subjected to affinity maturation. Isolated scFv were assessed for binding specificity and affinity and may provide the basis for the development of novel immunotherapeutic strategies. This work demonstrates the selection of a fully human antibody fragment from a patient-derived gene pool by in vitro ribosome display technology.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD28/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Linfoma no Hodgkin/inmunología , Biblioteca de Péptidos , Receptores de Antígenos de Linfocitos T/inmunología , Ribosomas/inmunología , Antígenos CD28/aislamiento & purificación , Humanos , Inmunoensayo/métodos , Linfoma no Hodgkin/patologíaRESUMEN
Changes in human DNA methylation patterns are an important feature of cancer development and progression and a potential role in other conditions such as atherosclerosis and autoimmune diseases (e.g., multiple sclerosis and lupus) is being recognised. The cancer genome is frequently characterised by hypermethylation of specific genes concurrently with an overall decrease in the level of 5 methyl cytosine. This hypomethylation of the genome largely affects the intergenic and intronic regions of the DNA, particularly repeat sequences and transposable elements, and is believed to result in chromosomal instability and increased mutation events. This review examines our understanding of the patterns of cancer-associated hypomethylation, and how recent advances in understanding of chromatin biology may help elucidate the mechanisms underlying repeat sequence demethylation. It also considers how global demethylation of repeat sequences including transposable elements and the site-specific hypomethylation of certain genes might contribute to the deleterious effects that ultimately result in the initiation and progression of cancer and other diseases. The use of hypomethylation of interspersed repeat sequences and genes as potential biomarkers in the early detection of tumors and their prognostic use in monitoring disease progression are also examined.
Asunto(s)
Metilación de ADN , Enfermedades Genéticas Congénitas/genética , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , Elementos Transponibles de ADN/fisiología , ADN Satélite/fisiología , Epigénesis Genética , Humanos , Elementos de Nucleótido Esparcido Largo/fisiología , Pronóstico , Secuencias Repetitivas de Ácidos Nucleicos , Retroelementos/fisiología , Elementos de Nucleótido Esparcido Corto/fisiologíaRESUMEN
IVC (in vitro compartmentalization) provides a complete cell-free approach for the production of novel targeted proteins. IVC uses aqueous droplets, which contain DNA and components for protein production, within water-in-oil emulsions. Recent advances in the composition and formation, as well as the detection, sorting and recovery, of the droplets enable the evolution of the encoded protein. Furthermore, IVC technology permits the step-wise addition of reagents into the droplets, making them suitable for high-throughput applications - where synthetic enzymes with substrate specificity are selected for catalytic activity, binding and regulation. In the broad field of in vitro display, developments such as the incorporation of unnatural amino acids and the production of cell toxic proteins expand the diverse spectrum of future applications for IVC.
Asunto(s)
Sistema Libre de Células/química , ADN/química , Evolución Molecular Dirigida , Diseño de Fármacos , Emulsiones/química , Ingeniería de Proteínas/métodos , Proteínas/síntesis química , Biomimética , Compartimento CelularRESUMEN
Display technologies are fundamental to the isolation of specific high-affinity binding proteins for diagnostic and therapeutic applications in cancer, neurodegenerative, and infectious diseases as well as autoimmune and inflammatory disorders. Applications extend into the broad field of antibody (Ab) engineering, synthetic enzymes, proteomics, and cell-free protein synthesis. Recently, in vitro display technologies have come to prominence due to the isolation of high-affinity human antibodies by phage display, the development of novel scaffolds for ribosome display, and the discovery of novel protein-protein interactions. In vitro display represents an emerging and innovative technology for the rapid isolation and evolution of high-affinity peptides and proteins. So far, only one clinical drug candidate produced by in vitro display technology has been approved by the FDA for use in humans, but several are in clinical or preclinical testing. This review highlights recent advances in various engineered biopharmaceutical products isolated by in vitro display with a focus on the commercial developments.
Asunto(s)
Biofarmacia/métodos , Técnicas Químicas Combinatorias , Ingeniería de Proteínas/métodos , Mapeo de Interacción de ProteínasRESUMEN
Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein-protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.
Asunto(s)
Terapia Biológica/métodos , Biblioteca de Péptidos , Proteínas Ribosómicas/química , Ribosomas/química , Animales , Anticuerpos , Membrana Celular/metabolismo , ADN Complementario/metabolismo , Evolución Molecular , Biblioteca de Genes , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/terapia , Enfermedades Neurodegenerativas/patología , Unión Proteica , Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
Engineered antibodies and their fragments are invaluable tools for a vast range of biotechnological and pharmaceutical applications. However, they are facing increasing competition from a new generation of protein display scaffolds, specifically selected for binding virtually any target. Some of them have already entered clinical trials. Most of these nonimmunoglobulin proteins are involved in natural binding events and have amazingly diverse origins, frameworks, and functions, including even intrinsic enzyme activity. In many respects, they are superior over antibody-derived affinity molecules and offer an ever-extending arsenal of tools for, e.g., affinity purification, protein microarray technology, bioimaging, enzyme inhibition, and potential drug delivery. As excellent supporting frameworks for the presentation of polypeptide libraries, they can be subjected to powerful in vitro or in vivo selection and evolution strategies, enabling the isolation of high-affinity binding reagents. This article reviews the generation of these novel binding reagents, describing validated and advanced alternative scaffolds as well as the most recent nonimmunoglobulin libraries. Characteristics of these protein scaffolds in terms of structural stability, tolerance to multiple substitutions, ease of expression, and subsequent applications as specific targeting molecules are discussed. Furthermore, this review shows the close linkage between these novel protein tools and the constantly developing display, selection, and evolution strategies using phage display, ribosome display, mRNA display, cell surface display, or IVC (in vitro compartmentalization). Here, we predict the important role of these novel binding reagents as a toolkit for biotechnological and biomedical applications.
Asunto(s)
Biblioteca de Péptidos , Proteínas/química , Proteínas/metabolismo , Animales , Técnicas Químicas Combinatorias , Humanos , Unión Proteica/fisiología , Mapeo de Interacción de Proteínas , Proteínas/aislamiento & purificaciónRESUMEN
In this study, human T cells were provided with a reactivity against the Lewis-Y (Le(Y)) carbohydrate antigen, which is overexpressed on 70% of epithelial-derived tumors, but not normally recognized by T cells. Antitumor reactivity was achieved by transduction of T cells with a gene encoding a cell-surface chimeric receptor composed of single-chain anti-Le(Y) antibody linked to an enhanced cytoplasmic signaling domain made up of CD28 and CD3-zeta. Importantly, the single-chain antibody was humanized to try to reduce potential problems of human anti-mouse antibody responses in patients receiving chimeric receptor-modified T cells in future clinical trials. T cells expressing the chimeric receptor were demonstrated to secrete cytokines and proliferate in response to receptor ligation and lysed Le(Y+) tumors in vitro. Another aspect of this study was the finding that no activity was observed against normal tissue, as represented by autologous neutrophils that express low levels of Le(Y). Significantly, systemic delivery of anti-Le(Y) T cells dramatically inhibited established s.c. human ovarian OVCAR-3 tumors (a recognized difficult model to treat) in mice. Finally, we demonstrated that anti-Le(Y) T cells preferentially expanded or accumulated in the tumor compared with control empty vector T cells, thereby providing mechanistic insight into the specific antitumor response. This study supports the use of humanized gene-modified T cells as a potential therapy for Le(Y+) malignancies.
Asunto(s)
Traslado Adoptivo , Inmunoterapia/métodos , Antígenos del Grupo Sanguíneo de Lewis/química , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/citología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Autoinmunidad , Antígenos CD28/biosíntesis , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Ensayos Clínicos como Asunto , Citocinas/metabolismo , Citometría de Flujo , Vectores Genéticos , Humanos , Inmunohistoquímica , Leucocitos Mononucleares/citología , Ratones , Trasplante de Neoplasias , Neoplasias/metabolismo , Neutrófilos/metabolismo , Proteínas Recombinantes de Fusión/química , Retroviridae/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.
Asunto(s)
Fragmentos de Inmunoglobulinas/inmunología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Fluorescencia , Humanos , Fragmentos de Inmunoglobulinas/genéticaRESUMEN
Diabodies (scFv dimers) are small, bivalent antibody mimetics of approximately 55kDa in size that possess rapid in vivo targeting pharmacokinetics compared to the intact parent antibody, and may prove highly suitable for imaging and therapeutic applications. Here, we describe T84.66Di, the first diabody crystal structure in which the scFvs comprise V domains linked in the V(L)-to-V(H) orientation. The structure was determined by X-ray diffraction analysis to 2.6 A resolution. The T84.66Di scFv was constructed from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66 variable domains connected by an eight residue peptide linker to provide flexibility between Fv modules and promote dimer formation with bivalent affinity to the cell-surface target, CEA. Therefore, it was surprising to observe a close association of some Fv module complementarity-determining regions in the T84.66 diabody crystal, especially compared to other diabody structures all of which are linked in the opposite V(H)-to-V(L) orientation. The differences between the arrangement of Fv modules in the T84.66Di V(L)-to-V(H) linked diabody structure compared to the crystal structure of L5MK16 and other proposed V(H)-to-V(L) linked diabodies has been investigated and their potential for flexibility discussed. The comparison between V(H)-to-V(L) and V(L)-to-V(H) linked diabodies revealed in this study represents a limited repertoire of possible diabody Fv orientations, but one that reveals the potential flexibility of these molecules. This analysis therefore provides some signposts that may impact on future molecular designs for these therapeutic molecules with respect to diabody flexibility and avidity.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígeno Carcinoembrionario/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Anticuerpos Biespecíficos , Reacciones Antígeno-Anticuerpo , Antígenos de Neoplasias/metabolismo , Cristalización , Cristalografía por Rayos X , Humanos , Fragmentos Fc de Inmunoglobulinas , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Isoenzimas/inmunología , Modelos Moleculares , Neuraminidasa/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosfolipasa C delta , Conformación Proteica , Proteínas Recombinantes de Fusión , Fosfolipasas de Tipo C/inmunologíaAsunto(s)
Fragmentos de Inmunoglobulinas/biosíntesis , Neoplasias/diagnóstico , Neoplasias/terapia , Reacción en Cadena de la Polimerasa/métodos , Anticuerpos/uso terapéutico , Cromatografía de Afinidad/métodos , Cartilla de ADN/química , Biblioteca de Genes , Ingeniería Genética/métodos , Humanos , Fragmentos de Inmunoglobulinas/uso terapéutico , Proteínas Recombinantes/uso terapéuticoRESUMEN
This protocol describes optimised large-scale bacterial fermentation conditions for recombinant single-chain Fv molecule (scFv) monomers and multimers (diabodies and triabodies). The heat-inducible bacterial secretion vector, pPOW3, utilising the temperature-regulated tandem lambda promoters is particularly suited to the large-scale fermentation of single-chain antibodies, providing low-cost recombinant protein synthesis. The protein expressed by this vector is secreted into the periplasm where it is found as both the soluble and insoluble protein that is associated with the cell membranes. A protein fractionation method for the rapid extraction and affinity purification of the soluble protein fraction and the urea solubilization and refolding of the insoluble protein fraction expressed from single-chain antibody (Ab) fragment gene constructs is described. This method is simple to perform and utilises inexpensive reagents to provide cost-effective protein synthesis.