Simon Krah

Single-domain antibodies for biomedical applications

Abstract Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope.

In this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1.

Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates.

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens.

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.

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.

Engineering bispecific antibodies with defined chain pairing

Bispecific IgG-like antibodies can simultaneously interact with two epitopes on the same or on different antigens. Therefore, these molecules facilitate novel modes of action, which cannot be addressed by conventional monospecific IgGs. However, the generation of such antibodies still appears to be demanding due to their specific architecture comprising four different polypeptide chains that need to assemble correctly. This review focusses on different strategies to circumvent this issue or to enforce a correct chain association with a focus on common-chain bispecific antibodies.

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

BackgroundYeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating.ResultsWithin this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties.ConclusionWe demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

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 Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries

Yeast surface display is a versatile platform technology for antibody discovery. Nevertheless, the construction of antibody Fab libraries typically is a tedious multistep process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Here, we present a focused one-step Golden Gate cloning approach for the generation of yeast surface display Fab libraries that allows for simultaneous introduction of heavy-chain and light-chain variable regions into one single display vector. Thereby, the overall time as well as the materials needed for library generation can be reduced significantly.

Construction of Histidine-Enriched Shark IgNAR Variable Domain Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments

The adaptive immune system of sharks comprises a heavy chain-only antibody isotype, referred to as immunoglobulin new antigen receptor (IgNAR). Antigen binding in case of IgNAR antibodies is mediated by a single variable domain (vNAR). Due to their inherent beneficial biophysical properties, such as small size and high thermal stability combined with a high specificity and affinity to their target antigens, vNAR domains emerged as promising tools for biotechnological and biomedical applications. Herein, we present detailed protocols for the engineering of pH-sensitivity into IgNAR V domains by constructing histidine-enriched and CDR3-diversified semisynthetic antibody libraries which can then be screened upon using yeast surface display. Protonation or deprotonation of incorporated histidine residues at different pH values results in structural transitions caused by altered electrostatic interactions. These interactions account for an altered binding behavior toward the target antigen. In the following protocol, we describe the generation of a semisynthetic vNAR master library that comprises two histidine residues on average in the 12-residue CDR3 loop. Moreover, once a pH-dependent vNAR population toward the target antigen is identified, this population can further be optimized in terms of affinity and pH sensitivity upon conducting a CDR1-mediated affinity maturation.