Phage display technology was first invented in 1985 for the display of polypeptides. Later, in 1990, the first antibody fragment was displayed on a phage. Since then, this technology has been successfully applied to discover hundreds of antibodies for research, diagnosis, and treatment, including over 14 clinically approved antibodies. Various aspects of phage display methods have been improved and advanced, enabling the discovery of antibodies targeting challenging targets and those with specific binding properties. Compared to other display technologies (such as ribosome, yeast, or mammalian/animal display), one advantage of phage display is the ability to create large libraries and store them for selection, which makes it possible to discover high-affinity antibodies against various antigens.

I.Antibodies used in phage display libraries

Phage display libraries can be designed using various phages, such as filamentous M13, fd, and f1 phages, to display different antibody formats. The two most commonly used formats are single-chain variable fragments (ScFvs) and antigen-binding fragments (ABFs). Other antibody formats are also employed to construct antibody phage display libraries, including human single-domain antibodies (human VH) and camel and shark single-domain antibodies (VHH and VNAR, respectively). VHHs are small (12-15 kDa) and consist solely of the antigen-binding fragment of the heavy chain antibody. Compared to conventional antibodies, the complementarity-determining region 3 (CDR3) loop in VHHs is typically elongated, enabling VHHs to bind antigens such as enzyme catalytic sites or receptor domains. VNAR fragments are similar in size to VHH antibody fragments, but a notable exception is that they have only two CDR loops due to the partial deletion of the Fr2-CDR2 region. The choice of antibody format for phage display activities depends on the intended application of the discovered antibody. If the application is therapeutic and has a long half-life, scFv or Fab libraries may be the optimal choice, as they can be easily reformatted into the commonly used therapeutic IgG format. For research reagents and diagnostic applications, or when the cost of large-scale production is a major concern, formats like VHH may be the most ideal. In summary, describing the requirements for the final antibody product is crucial to selecting the most appropriate library type.

II.Antigen presentation strategy

For a successful phage display-based antibody discovery project, it is crucial that the conformation of the included antigens resembles their conformation in the final application. Otherwise, the discovered antibodies may ultimately only recognize antigens with altered conformation. Therefore, the initial and critical step in phage display activities is to determine the optimal antigen presentation strategy. The most widely used antigen presentation strategy involves direct or indirect immobilization of antigens on surfaces. In direct immobilization, antigens are coated onto surfaces via passive adsorption. This strategy remains the simplest antigen presentation method to date; however, it is less suitable for many types of antigens that undergo conformational changes during adsorption. The issue may be more pronounced for small antigens that may lack sufficient intermolecular attraction for passive adsorption. For some antigens, indirect immobilization can be employed as an alternative to direct immobilization.

Through indirect immobilization, antigens are captured on surfaces using capture molecules. The most prevalent technique leverages the strong binding between streptavidin/avidin and biotin, where streptavidin/avidin coats the surface, and antigens are conjugated to biotin via ligands or tags. This enables indirect and stable attachment of antigens to the surface. Indirect immobilization is more likely to preserve the native conformation of antigens; however, it is crucial to avoid excessive biotinylation, as this may mask important epitopes or lead to antigen aggregation. Two distinct biotinylation strategies exist: site-specific biotinylation and random biotinylation. Site-specific biotinylation can be achieved using biotinylating receptor peptides (BAPs), including enzyme biotinylation sites. AviTag is one of the most widely used BAPs, requiring recombinant expression of the target antigen fused to a 15-amino acid peptide tag. The lysine residues of the Av32iTag sequence are biotinylated by the E. coli biotin ligase BirA. Av33iTag-anchored antigens can be co-expressed with BirA in bacterial, yeast, and mammalian cells to achieve in vivo biotinylation. Alternatively, purified Avi-tagged antigens can be incubated with purified BirA and biotin for in vitro biotinylation. BAPs-mediated biotinylation results in the addition of a single biotin at each antigen site, thereby controlling the antigen-to-biotin ratio and preventing excessive biotinylation. However, the AviTag system cannot be used in all scenarios, particularly when recombinant expression of the target antigen is difficult or impractical, or when AviTag may interfere with the potentially important (terminal) epitopes of the antigen.

 As an alternative to BAP biotinylation, randomized chemical biotinylation can be employed. In this method, purified antigens and biotinylating reagents are mixed with various potential reaction chemicals to achieve covalent linkage between antigens and biotin. Multiple linkers are available, enabling biotinylation of antigens on primary amines (N-terminus or side chains of lysine residues) or thiol and carboxyl groups. Although faster and more cost-effective than enzymatic biotinylation (via E. coli biotin ligase), chemical biotinylation requires titration to achieve the desired 1:1 antigen-to-biotin ratio. Indirect immobilization of antigens can also be utilized depending on the peptide tag and capture molecule specificity. This necessitates recombinant expression of antigens in fusion proteins, coated with peptide tags and selective surface capture molecules. The binding between the tag and capture molecule leads to antigen immobilization. Although less prevalent than the biotin-lectin system, His tags and anti-His antibodies or other His capture molecules have been employed for antigen presentation in phage display selection. In recent years, a peptide-protein ligand called SpyTag/SpyCatcher has been isolated from Streptococcus pyogenes fibroconnexin-binding protein and has been utilized for antigen presentation in phage display selection. The binding between Spy-Tag and SpyCatcher occurs via an isopeptide bond and is reported to be irreversible, specific, and highly adaptable to various conditions (e.g., pH, temperature, and buffer).

III.Application of Phage Library

 Phage display antibody libraries are widely used in screening high-affinity antibodies, such as those targeting angiogenesis markers like fibronectin, tumor-specific B3/B4 antigens, and EGFR. These libraries are typically constructed through phage display technology, which involves RT-PCR amplification of mRNA from peripheral blood mononuclear cells, spleen cells, bone marrow cells, and tonsils to generate cDNA fragments. These gene fragments are then spliced into phage-like particles, which are transferred into phages to form covalently linked structural proteins for expression and in vitro display. The heavy and light chains of the antibodies are expressed independently and randomly combined, resulting in an extensive antibody library with phage clones numbering between 10^11 and 10^12.

KMD Bioscience has established a comprehensive and mature phage antibody display (phage display) technology platform. Based on this platform, KMD Bioscience can provide key experimental services including antigen design, alpaca immunization, library construction and screening, as well as activity and function validation, offering high-specificity and high-affinity alpaca VHH antibodies to scientists worldwide. Additionally, KMD Bioscience possesses extensive experience in antibody engineering and can provide integrated upstream and downstream antibody services, such as antibody humanization, human scFV antibody library construction, human Fab antibody library construction, human antibody phage library preparation, phosphorylation antibody customization, and antibody affinity maturation services, to meet the diverse research needs of clients.

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