Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Isolation of Anti-Hapten Antibodies by Fluorescence-Activated Cell Sorting of Yeast-Displayed B-Cell Receptor Gene Repertoires.

Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.

Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries.

In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species' cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.

Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display.

Fluorescence-activated cell sorting (FACS) in combination with yeast surface display (YSD) has proven to be a valuable tool for the engineering of antibodies. It enables the fast and robust identification and isolation of candidates with prescribed characteristics from combinatorial libraries. A novel application for FACS and YSD that has recently evolved addresses the engineering of antibodies toward pH-switchable antigen binding, aiming at reduced binding at acidic pH, compared to neutral pH. Therefore, we give guidance for the incorporation of such pH switches into antibody variable domains using combinatorial histidine scanning libraries. The protocol describes a flow cytometric sorting technique for the enrichment of antigen-specific molecules. Moreover, we provide information on how to screen the obtained antibody pools from initial sorting to isolate and characterize pH-sensitive variants.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.