RESUMEN
The use of next generation sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.
Asunto(s)
Anticuerpos/química , Bacteriófagos/genética , Descubrimiento de Drogas , Secuenciación de Nucleótidos de Alto Rendimiento , Biblioteca de PéptidosRESUMEN
Phage display has emerged as one of the leading technologies for the selection of highly specific monoclonal antibodies, offering a number of advantages over traditional methods of antibody generation. While there are various possibilities to conduct phage display (e.g., solution panning, solid-phase panning), whole cell panning is an elegant way to present membrane embedded target antigens in their natural environment and conformation to antibody-bearing phages. Here, a whole cell panning procedure using a Fab-based antibody library including primary cell based screening for selectivity is described.
Asunto(s)
Antígenos/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Membrana Celular/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de PéptidosRESUMEN
pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.
Asunto(s)
Quimiocina CXCL10/química , Ingeniería de Proteínas , Anticuerpos de Cadena Única/química , Quimiocina CXCL10/genética , Humanos , Concentración de Iones de Hidrógeno , Anticuerpos de Cadena Única/genéticaRESUMEN
The plasticity of natural immunoglobulin repertoires can be exploited for the generation of phage display libraries. Secondary lymphoid organs, such as the spleen and the lymph nodes, constitute interesting sources of diversity because they are rich in B cells, part of which can be affinity matured. These organs, however, differ in their anatomical structure, reflecting the different fluids they drain, which affects the B cell repertoires. The CDRH3 repertoires from these organs, extracted from naïve or immunized mice, were compared in the context of phage display libraries using human antibody framework families. Deep sequencing analysis revealed that all libraries displayed different CDRH3 repertoires, but the one derived from lymph nodes of naïve mice was the most diverse. Library performance was assessed by in vitro selection. For both organs, immunization increased substantially the frequency of molecules able to bind to the immunogen. The library derived from lymph nodes from naïve mice, however, was the most effective in generating diverse and high affinity candidates. These results illustrate that the use of a biased CDRH3 repertoire increases the performance of libraries, but reduces the clonal diversity, which may be detrimental for certain strategies.
Asunto(s)
Anticuerpos/inmunología , Linfocitos B/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Diversidad de Anticuerpos/genética , Diversidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/metabolismo , Quimiocina CCL5/inmunología , Quimiocina CCL5/metabolismo , Regiones Determinantes de Complementariedad/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, ß2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores Fc/genética , Receptores Fc/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Biotinilación/métodos , Células Cultivadas , Cricetinae , ADN Complementario/genética , Vectores Genéticos/genética , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Inmunoglobulina G/inmunología , Cinética , Ratones , Unión Proteica , Receptores Fc/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Resonancia por Plasmón de Superficie/métodosRESUMEN
Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.