Antibody Selection via Phage Display in Microtiter Plates.

The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection ("panning"), screening, and identification of monoclonal antibodies in less than 2 weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates.

Antibody Batch Cloning.

The antigen-binding ability of each antibody clone selected by phage display is usually initially ranked by a screening ELISA using monovalent scFv antibody fragments. Further characterization often requires bivalent antibody molecules such as IgG or scFv-Fc fusions. To produce these, the V region encoding genes of selected hits have to be cloned into a mammalian expression vector and analyzed as a bivalent molecule, requiring a laborious cloning procedure. We established a high-throughput procedure allowing rapid screening of candidates in bivalent formats. This protocol allows for the parallelized cloning of all selected antibody fragments into a mammalian expression vector in the 96-well plate format. The bivalent antibody molecules can then be produced and purified in 96-well plates for further analysis in microtiter plate assays.

Mapping Epitopes by Phage Display.

Monoclonal antibodies (mAbs) are valuable biological molecules, serving for many applications. Therefore, it is advantageous to know the interaction pattern between antibodies and their antigens. Regions on the antigen which are recognized by the antibodies are called epitopes, and the respective molecular counterpart of the epitope on the mAbs is called paratope. These epitopes can have many different compositions and/or structures. Knowing the epitope is a valuable information for the development or improvement of biological products, e.g., diagnostic assays, therapeutic mAbs, and vaccines, as well as for the elucidation of immune responses. Most of the techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Among the techniques used for epitope mapping, phage display is a versatile technology that allows the display of a library of oligopeptides or fragments from a single gene product on the phage surface, which then can interact with several antibodies to define epitopes. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

Generation of Chimeric Antigen Receptors against Tetraspanin 7.

Adoptive transfer of antigen-specific regulatory T cells (Tregs) has shown promising results in the treatment of autoimmune diseases; however, the use of polyspecific Tregs has limited effects. However, obtaining a sufficient number of antigen-specific Tregs from patients with autoimmune disorders remains challenging. Chimeric antigen receptors (CARs) provide an alternative source of T cells for novel immunotherapies that redirect T cells independently of the MHC. In this study, we aimed to generate antibody-like single-chain variable fragments (scFv) and subsequent CARs against tetraspanin 7 (TSPAN7), a membrane protein highly expressed on the surface of pancreatic beta cells, using phage display technology. We established two methods for generating scFvs against TSPAN7 and other target structures. Moreover, we established novel assays to analyze and quantify their binding abilities. The resulting CARs were functional and activated specifically by the target structure, but could not recognize TSPAN7 on the surface of beta cells. Despite this, this study demonstrates that CAR technology is a powerful tool for generating antigen-specific T cells and provides new approaches for generating functional CARs.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy.

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

Baculovirus-free insect cell expression system for high yield antibody and antigen production.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

Epitope Mapping via Phage Display from Single-Gene Libraries.

Antibodies are widely used in a large variety of research applications, for diagnostics and therapy of numerous diseases, primarily cancer and autoimmune diseases. Antibodies are binding specifically to target structures (antigens). The antigen-binding properties are not only dependent on the antibody sequence, but also on the discrete antigen region recognized by the antibody (epitope). Knowing the epitope is valuable information for the improvement of diagnostic assays or therapeutic antibodies, as well as to understand the immune response of a vaccine. While huge progress has been made in the pipelines for the generation and functional characterization of antibodies, the available technologies for epitope mapping are still lacking effectiveness in terms of time and effort. Also, no technique available offers the absolute guarantee of succeeding. Thus, research to develop and improve epitope mapping techniques is still an active field. Phage display from random peptide libraries or single-gene libraries are currently among the most exploited methods for epitope mapping. The first is based on the generation of mimotopes and it is fastened to the need of high-throughput sequencing and complex bioinformatic analysis. The second provides original epitope sequences without requiring complex analysis or expensive techniques, but depends on further investigation to define the functional amino acids within the epitope. In this book chapter, we describe how to perform epitope mapping by antigen fragment phage display from single-gene antigen libraries and how to construct these types of libraries. Thus, we also provide figures and analysis to demonstrate the actual potential of this technique and to prove the necessity of certain procedural steps.