Isolation of Antigen-Specific VHH Single-Domain Antibodies by Combining Animal Immunization with Yeast Surface Display.

In addition to conventional hetero-tetrameric antibodies, the adaptive immune repertoire of camelids comprises the so-called heavy chain-only antibodies devoid of light chains. Consequently, antigen binding is mediated solely by the variable domain of the heavy chain, referred to as VHH. In recent years, these single-domain moieties emerged as promising tools for biotechnological and biomedical applications. In this chapter, we describe the generation of VHH antibody yeast surface display libraries from immunized Alpacas and Lamas as well as the facile isolation of antigen-specific molecules in a convenient fluorescence-activated cell sorting (FACS)-based selection process.

Chemical Modification of the Yeast Cell Surface Allows the Switch Between Display and Soluble Secretion of Full-Length Antibodies.

The classical yeast surface display (YSD)-based antibody hit discovery relies on the enrichment of candidates from large antibody fragment libraries by fluorescence-activated cell sorting (FACS), followed by sequencing of the remaining diversity. The sequences of hit candidates are then transferred into plasmids, which allow their expression as full-length IgG in mammalian cells. After production and downstream processing, antibodies are further qualified in biochemical and functional assays. This multistep process has been proven to be successful in academia and has also found its way to the biopharmaceutical industry. However, during the recent decade, several groups tried to simplify this process by enabling either switchable or simultaneous secretion and display of antibodies or antibody fragments by yeast cells. Therefore, functional testing of candidates can be accomplished immediately during the screening process, while eliminating the need for tedious sequence reformatting and production host change. In this protocol, we give guidance for the establishment of a full-length antibody display system on yeast cells, which permits a simple switch to soluble antibody secretion.

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.

Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display.

Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display. bsAbs with subnanomolar affinities were identified, wherein each separate binding arm mediated specific binding to the respective antigen. Altogether, the described strategy represents a combination of in vivo immunization with an in vitro selection method, which allows for the integration of existing therapeutic antibodies into a bispecific format.

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.

REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.