RESUMEN
A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.
Asunto(s)
Bacteriófago M13/genética , Regiones Determinantes de Complementariedad/genética , Fragmentos Fab de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Oligosacáridos/genética , Oligosacáridos/inmunología , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Diversidad de Anticuerpos , Bacteriófago M13/química , Bacteriófago M13/inmunología , Sitios de Unión de Anticuerpos , Secuencia de Carbohidratos , Regiones Determinantes de Complementariedad/química , Diseño de Fármacos , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/química , Antígenos del Grupo Sanguíneo de Lewis , Ratones , Datos de Secuencia Molecular , Oligosacáridos/química , Electricidad EstáticaRESUMEN
Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.
Asunto(s)
Pared Celular/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Afinidad de Anticuerpos , Pared Celular/inmunología , Clonación Molecular , Diploidia , Citometría de Flujo/métodos , Vectores Genéticos/genética , Haploidia , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Mutación , Biblioteca de Péptidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/aislamiento & purificación , Estreptavidina/inmunologíaRESUMEN
We introduce a procedure for the rapid generation of fully human antibodies derived from "Fab-on-phage" display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented. The average transient expression levels of the IgG constructs in HEK293T cells are above 10 microg/ml, allowing the use of conditioned media in functional assays without antibody purification. Furthermore, we describe a high-throughput purification method yielding IgG amounts sufficient for initial antibody characterization. Our technology allows the generation and production of antigen-specific complete human antibodies as fast or even faster than raising monoclonal antibodies by conventional hybridoma techniques.