ABSTRACT
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.
Subject(s)
Bacteriophage M13/genetics , Complementarity Determining Regions/genetics , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Heavy Chains/genetics , Oligosaccharides/genetics , Oligosaccharides/immunology , Peptide Library , Protein Engineering/methods , Amino Acid Sequence , Animals , Antibody Diversity , Bacteriophage M13/chemistry , Bacteriophage M13/immunology , Binding Sites, Antibody , Carbohydrate Sequence , Complementarity Determining Regions/chemistry , Drug Design , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Heavy Chains/chemistry , Lewis Blood Group Antigens , Mice , Molecular Sequence Data , Oligosaccharides/chemistry , Static ElectricityABSTRACT
Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.
Subject(s)
Antibody Affinity , Antibody Formation , Complementarity Determining Regions/genetics , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/immunology , Peptide Library , Protein Engineering/methods , Genetic Variation/genetics , Humans , Immunoglobulin Fab Fragments/genetics , Protein Binding , Recombination, Genetic/genetics , Tissue DonorsABSTRACT
Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.
Subject(s)
Immunoglobulin Fab Fragments/immunology , Receptor, TIE-1/immunology , Antibody Affinity/immunology , Chromatography, Affinity , Escherichia coli/genetics , Humans , Immunoglobulin Fab Fragments/analysis , Immunoglobulin Fab Fragments/genetics , Kinetics , Peptide Library , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Staphylococcal Protein A , Surface Plasmon Resonance/methodsABSTRACT
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.
Subject(s)
Cell Wall/metabolism , Immunoglobulin Fab Fragments/metabolism , Saccharomyces cerevisiae/metabolism , Antibody Affinity , Cell Wall/immunology , Cloning, Molecular , Diploidy , Flow Cytometry/methods , Genetic Vectors/genetics , Haploidy , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Mutation , Peptide Library , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/isolation & purification , Streptavidin/immunologyABSTRACT
We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.
Subject(s)
Antibody Affinity , Flow Cytometry/methods , Immunoglobulin Fab Fragments/metabolism , Saccharomyces cerevisiae/isolation & purification , Fluorescence , Genetic Vectors , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Streptavidin/metabolismABSTRACT
In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.