RESUMO
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
Assuntos
Escorpiões , Peçonhas , Animais , Humanos , Escorpiões/química , Peçonhas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismoRESUMO
Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.
Assuntos
Proteínas de Bactérias/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Polissacarídeos Bacterianos/deficiência , Infecções por Rotavirus/microbiologia , Rotavirus/fisiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Lacticaseibacillus rhamnosus/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Probióticos/administração & dosagem , Infecções por Rotavirus/prevenção & controle , Infecções por Rotavirus/virologiaRESUMO
Since genetic engineering of humanized murine monoclonal antibodies was first demonstrated over two decades ago, antibody engineering technologies have evolved based upon an increasing understanding of the mechanisms involved in antibody generation in vivo, and a constant search for alternative routes to evolve and exploit the characteristics of antibodies. As a result, antibody engineers have devised innovative strategies for the rapid evolution and selection of antibodies and novel antibody designs (i.e., antibody fragments). Phage display, cell display and ribosome display technologies, which comprise the core of the currently available technologies for the discovery and preparation of such antibodies, are reviewed herein. This article intends to communicate the state-of-the-art technology available for the engineering of antibodies to a general readership interested in this important field. Therefore, important immunology concepts are introduced before detailed descriptions of the three antibody engineering technologies are presented in later sections. A comparison of these methodologies suggests that despite the predominance of phage display for the engineering of antibody fragments in the past 20 years, cell display and ribosome display will likely gain importance in the selection and discovery of the antibody fragments in the future. Finally, these technologies are likely to play an important role in the production of the next generation of antibody-based therapeutics.
Las tecnologías para la ingeniería de anticuerpos han evolucionado durante las últimas dos décadas, desde la demostración de la posibilidad de humanizar anticuerpos monoclonales de ratón mediante ingeniería genética, apoyadas en el creciente entendimiento de los mecanismos involucrados en la generación de anticuerpos in vivo, y en una búsqueda constante de rutas alternativas para evolucionar y explotar sus características. Es así como los ingenieros de anticuerpos han desarrollado estrategias innovadoras para la evolución y selección de anticuerpos y de novedosos diseños de anticuerpos conocidos como fragmentos de anticuerpos. Esta revisión se enfoca en tres tecnologías que comprenden el núcleo de las tecnologías actualmente disponibles para el descubrimiento y preparación de tales anticuerpos: la presentación en fagos, la presentación en células, y la presentación en ribosomas. Este artículo busca presentar el estado del arte de estas tecnologías a un grupo general de lectores interesados en este campo, por lo que inicialmente se introducen importantes conceptos de inmunología requeridos para comprender en detalle las tecnologías discutidas. Una comparación de estas metodologías para la ingeniería de anticuerpos sugiere que a pesar del dominio de las tecnologías basadas en la presentación en fagos durante los últimos 20 años, en los próximos años la presentación en células y la presentación en ribosomas probablemente ganarán importancia para la selección y descubrimiento de fragmentos de anticuerpos. Finalmente, es probable que estas tecnologías jueguen un papel importante en la producción de la siguiente generación de terapéuticos basados en anticuerpos.
Assuntos
Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Engenharia de Proteínas/tendênciasRESUMO
Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than OKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with OKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity.
Assuntos
Doenças Autoimunes/terapia , Complexo CD3/imunologia , Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto/terapia , Imunoterapia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Doenças Autoimunes/imunologia , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia Genética , Rejeição de Enxerto/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Muromonab-CD3/farmacologia , Transplante de Órgãos , Proteínas Recombinantes de Fusão , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Antibody fragments to the four Dengue virus serotypes were isolated from a human universal naïve library using phage display technology. Phage-displayed antibody fragments were selected on Dengue virus particles directly captured from infected Vero cells supernatant by an anti-dengue monoclonal antibody, in order to avoid laborious virus concentration/purification procedures. A total of nine phage-displayed antibody fragments were obtained. Seven of them were highly specific for three of the selector serotypes (two for Dengue 1, four for Dengue 3 and one for Dengue 4). One clone (Dengue 3-selected) cross-reacted with Dengue 1, whereas another (selected with Dengue 2) cross-reacted with the three remaining serotypes. The soluble variants of six antibody fragments recognized their target viruses when used at nanomolar and even subnanomolar concentrations. All phage-displayed antibody fragments were cross-reactive against several strains of distinct genotypes within the corresponding serotype(s). These antibody fragments are potentially useful for the future development of tools for viral diagnosis and serotype identification. The simple phage selection method on captured virus could be applied in a high throughput way to obtain larger panels of antibody fragments to Dengue virus for multiple applications.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Fragmentos de Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Animais , Anticorpos Antivirais/genética , Especificidade de Anticorpos , Bacteriófagos , Linhagem Celular , Reações Cruzadas , Vírus da Dengue/classificação , Humanos , Fragmentos de Imunoglobulinas/genética , SorotipagemRESUMO
A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.
Assuntos
Bacteriófago M13/genética , Histoplasma/genética , Histoplasma/imunologia , Fragmentos de Imunoglobulinas/imunologia , Bacteriófago M13/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Biblioteca de PeptídeosRESUMO
Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA.
Assuntos
Adenocarcinoma/imunologia , Anticorpos Anti-Idiotípicos/genética , Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/imunologia , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mimetismo Molecular/imunologia , Adenocarcinoma/patologia , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos , Neoplasias do Colo/patologia , Feminino , Humanos , Hibridomas/imunologia , Imunização , Fragmentos de Imunoglobulinas/administração & dosagem , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/administração & dosagem , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.
Assuntos
Região Variável de Imunoglobulina/genética , Biblioteca de Peptídeos , Sequência de Aminoácidos , Antígenos/metabolismo , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Linfócitos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Phage display was introduced almost 20 years ago. It has been used to produce large amounts of diverse proteins, to analyze protein-ligand interactions, to improve the affinity of proteins for their binding receptors, and to characterize antibody binding sites. The recombinant version of the antibody Fv is termed single-chain variable fragment (scFv). Many large phage libraries have been developed that have yielded antibodies to several hundred antigens, but only 5 human anti-beta2-glycoprotein-I and three anti-prothrombin antibodies in scFV have been so far characterized. Antibodies to beta2GP-I thus generated show 92-94% homology with their nearest germ line genes. Their mutations frequently appear to be independent of antigen. Two anti-prothrombin antibodies show strong crossreactivity with beta2GP-I. Four mouse anti-beta2GP-I scFV show less binding properties than their original counterparts, but had the same capacity of inducing experimental antiphospholipid syndrome. This pathogenicity appears to reside in the V(H)DJ(H)C(H) region of the scFv since the V(H)DJ(H)C(H) regions of pathogenic scFV combined with irrelevant V(L) J(L)C(L) regions retained their pathogenicity while the opposite failed to do so.
Assuntos
Autoanticorpos/química , Glicoproteínas/imunologia , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Animais , Autoanticorpos/biossíntese , Autoanticorpos/genética , Autoanticorpos/fisiologia , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/fisiologia , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/fisiologia , Camundongos , Biblioteca de Peptídeos , beta 2-Glicoproteína IRESUMO
This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.