Antibody display technologies: selecting the cream of the crop.

Antibody display technologies enable the successful isolation of antigen-specific antibodies with therapeutic potential. The key feature that facilitates the selection of an antibody with prescribed properties is the coupling of the protein variant to its genetic information and is referred to as genotype phenotype coupling. There are several different platform technologies based on prokaryotic organisms as well as strategies employing higher eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic antibodies approved for therapy that have been discovered or engineered using this approach. In recent years several other technologies gained a certain level of maturity, most strikingly mammalian display. In this review, we delineate the most important selection systems with respect to antibody generation with an emphasis on recent developments.

Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning.

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

Cell Line Development Using Targeted Gene Integration into MAR-Rich Landing Pads for Stable Expression of Transgenes.

Integration of a gene of interest (GOI) into the genome of mammalian cells is the first step of cell line development campaigns for the production of biotherapeutics. Besides random integration methods, targeted gene integration approaches have emerged as promising tools over the last few years. In addition to reducing heterogeneity within a pool of recombinant transfectants, this process can also facilitate shorter timelines of the current cell line development process. Herein, we describe protocols for generating host cell lines carrying matrix attachment region (MAR)-rich landing pads (LPs), including BxB1 recombination sites. These LP-containing cell lines allow for site-specific and simultaneous integration of multiple GOIs. The resulting transgene-expressing stable recombinant clones can be used for the production of mono- or multispecific antibodies.

Generation of a host cell line containing a MAR-rich landing pad for site-specific integration and expression of transgenes.

In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix-Attachment Region (MAR)-rich landing pads (LPs), which allow for the simultaneous and site-specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR-based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP-containing host cell lines can be used for the site-specific integration of several GOIs and thus, generation of transgene-expressing stable recombinant clones. Transgene expression was shown by site-specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi-specific antibody (bsAb). The genetic stability of the herein described LP-based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR-rich landing pads can be successfully used for stable expression of one or several GOIs.

Humanization of Chicken-Derived scFv Using Yeast Surface Display and NGS Data Mining.

Generation of high-affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken-derived antibodies. To this end, high-affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high-affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high-affinity binders in the scFv and Fab formats. Next-generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high-throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.

Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays.

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

Simplifying the Detection of Surface Presentation Levels in Yeast Surface Display by Intracellular tGFP Expression.

Yeast surface display (YSD) is an ultra-high throughput method used in protein engineering. Protein-protein interactions as well as surface presentation on the yeast cell surface are verified through fluorophore-conjugated labeling agents.In this chapter we describe an improved setup for full-length surface presentation detection. To this end, we used a single open reading frame (ORF) encoding for the protein to be displayed and a 2A sequence and tGFP for an intracellular fluorescence signal. The 2A sequence allows the simultaneous generation of two separate proteins from the same ORF through ribosomal skipping. The entangled expression of the POI on the yeast surface and intracellular tGFP obviates the need for fluorescent staining steps.

Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform.

Here, we report the characterization of a VHH-derived IgG-like bi- and trispecific antibody platform that essentially relies on the replacement of the VH and VL regions of a conventional antibody by two independently functioning VHH domains. Consequently, a VHH is engrafted onto constant region CH1 while the other VHH-based paratope is engrafted on the constant region of the light chain, Cκ or Cλ, resulting in a tetravalent bispecific IgG-like molecule. Combined with a heavy chain heterodimerization technique, this platform allows facile engineering of bi- and trispecific antibodies with flexible valencies. We demonstrate the general applicability of this generic platform approach and elaborate on the limitations of specific formats.

A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies.

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8-3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl2 exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.

Dual Function pH Responsive Bispecific Antibodies for Tumor Targeting and Antigen Depletion in Plasma.

Shedding of membrane-bound cell surface proteins, where the extracellular domain is released and found in the circulation is a common phenomenon. A prominent example is CEACAM5 (CEA, CD66e), where the shed domain plays a pivotal role in tumor progression and metastasis. For treatment of solid tumors, the presence of the tumor-specific antigen in the plasma can be problematic since tumor-specific antibodies might be intercepted by the soluble antigen before invading their desired tumor target area. To overcome this problem, we developed a generic procedure to generate bispecific antibodies, where one arm binds the antigen in a pH-dependent manner thereby enhancing antigen clearance upon endosomal uptake, while the other arm is able to target tumor cells pH-independently. This was achieved by incorporating pH-sensitive binding modalities in the common light chain IGKV3-15*01 of a CEACAM5 binding heavy chain only antibody. Screening of a histidine-doped light chain library using yeast surface display enabled the isolation of pH-dependent binders. When such a light chain was utilized as a common light chain in a bispecific antibody format, only the respective heavy/light chain combination, identified during selections, displayed pH-responsive binding. In addition, we found that the altered common light chain does not negatively impact the affinity of other heavy chain only binders toward their respective antigen. Our strategy may open new avenues for the generation of bispecifics, where one arm efficiently removes a shed antigen from the circulation while the other arm targets a tumor marker in a pH-independent manner.

Yeast Surface Display in Combination with Fluorescence-activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens.

Yeast surface display emerged as a viable tool for the generation of human and murine monoclonal antibodies. This platform technology enables the careful definition of selection conditions, the potential for high-throughput screening, as well as the isolation of antibodies recognizing predefined epitopes. In this study, the applicability of yeast surface display in combination with fluorescence-activated cell sorting (FACS) for the isolation of antigen-specific chicken-derived antibodies is demonstrated. To this end, yeast-displayed recombinant antibody libraries from splenic mRNA of chickens immunized with epidermal growth factor receptor (EGFR) and human chorionic gonadotropin (hCG) were constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction. A large number of antigen binding scFvs were readily isolated in a convenient screening process. Target-specific scFv-Fc molecules were produced as soluble proteins and more extensively characterized by confirming specificity, thermostability and high affinity. Essentially, we demonstrated the biotechnological applicability of binders directed against both antigens via specific cellular binding for EGFR and in the context of a lateral flow test by utilizing hCG-binding scFvs as capturing antibodies for pregnancy detection. Altogether, the described strategy using yeast surface display expands the repertoire of display methods for the isolation of antibodies resulting from chicken immunization campaigns.

Generation of Semi-Synthetic Shark IgNAR Single-Domain Antibody Libraries.

Besides classical antibodies with the composition of heavy and light chains, sharks produce a unique heavy chain only isotype, termed Immunoglobulin New Antigen Receptor (IgNAR), in which antigen binding is solely mediated by a single domain, referred to as vNAR. Owing to their high affinity and specificity combined with their small size and high stability, vNAR domains emerged as promising target-binding scaffolds that can be tailor-made for biotechnological and biomedical applications. Herein, we describe protocols for the construction of semi-synthetic, CDR3-randomized vNAR libraries for the isolation of target-specific antibodies using yeast surface display or phage display as platform technology. Additionally, we provide information for affinity maturation of target-specific molecules through CDR1 diversification and sublibrary establishment.

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.